Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size.

BACKGROUND: The activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified. OBJECTIVES: To develop an assay that can differentiate the platelet-binding activity of VWF multimers. METHODS: A novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers. RESULTS: Of the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab6C11 to that of HMWMs, which is significantly higher than all other activity assays tested. CONCLUSION: The VWF:GPIbNab6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.

Haemophilia in the era of novel therapies: Where do inhibitors feature in the new landscape?

INTRODUCTION: The advent of therapeutic recombinant factor VIII (FVIII) and factor IX (FIX) protein infusions revolutionized the care of persons with haemophilia in the 1990s. It kicked off an era with the increasing use of prophylactic factor infusions for patients and transformed conversations around the ideal trough activity levels as well as the ultimate goals in tailored, individualized care. Our knowledge surrounding the immunologic basis of inhibitor development and treatment derives from a time when patients were receiving frequent factor infusions and focused on immune tolerance induction following inhibitor development. DISCUSSION: More recently, care was revolutionized again in haemophilia A with the approval of emicizumab, a bispecific antibody mimicking activated FVIII function, to prevent bleeding. The use of emicizumab prophylaxis has resulted in a significantly slower accumulation of factor exposure days and continued effective prophylaxis in the case of inhibitor development. While emicizumab is effective at reducing the frequency of bleeding events in patients with haemophilia A, management of breakthrough bleeds, trauma, and surgeries still requires additional treatment. Ensuring that FVIII is a therapeutic option, particularly for life-threatening bleeding events and major surgeries is critical to optimizing the care of persons with haemophilia A. Other novel non-factor concentrate therapies, including rebalancing agents, will dramatically change the landscape for persons with haemophilia B with inhibitors. CONCLUSION: This review discusses the changing landscape regarding the timing of inhibitor development and management strategies after inhibitor development, stressing the importance of education across the community to continue to vigilantly monitor for inhibitors and be prepared to treat persons with inhibitors.

Factor VIII antibody immune complexes modulate the humoral response to factor VIII in an epitope-dependent manner.

Introduction: Soluble antigens complexed with immunoglobulin G (IgG) antibodies can induce robust adaptive immune responses in vitro and in animal models of disease. Factor VIII immune complexes (FVIII-ICs) have been detected in individuals with hemophilia A and severe von Willebrand disease following FVIII infusions. Yet, it is unclear if and how FVIII-ICs affect antibody development over time. Methods: In this study, we analyzed internalization of FVIII complexed with epitope-mapped FVIII-specific IgG monoclonal antibodies (MAbs) by murine bone marrow-derived dendritic cells (BMDCs) in vitro and antibody development in hemophilia A (FVIII-/-) mice injected with FVIII-IC over time. Results: FVIII complexed with 2-116 (A1 domain MAb), 2-113 (A3 domain MAb), and I55 (C2 domain MAb) significantly increased FVIII uptake by BMDC but only FVIII/2-116 enhanced antibody titers in FVIII-/- mice compared to FVIII alone. FVIII/4A4 (A2 domain MAb) showed similar FVIII uptake by BMDC to that of isolated FVIII yet significantly increased antibody titers when injected in FVIII-/- mice. Enhanced antibody responses observed with FVIII/2-116 and FVIII/4A4 complexes in vivo were abrogated in the absence of the FVIII carrier protein von Willebrand factor. Conclusion: These findings suggest that a subset of FVIII-IC modulates the humoral response to FVIII in an epitope-dependent manner, which may provide insight into the antibody response observed in some patients with hemophilia A.

Qualification of Hemophilia Treatment Centers to Enable Multi-Center Studies of Gene Expression Signatures in Blood Cells from Pediatric Patients.

Hemophilia A is a rare congenital bleeding disorder caused by a deficiency of functionally active coagulation factor VIII (FVIII). Most patients with the severe form of the disease require FVIII replacement therapies, which are often associated with the development of neutralizing antibodies against FVIII. Why some patients develop neutralizing antibodies while others do not is not fully understood. Previously, we could demonstrate that the analysis of FVIII-induced gene expression signatures in peripheral blood mononuclear cells (PBMC) obtained from patients exposed to FVIII replacement therapies provides novel insights into underlying immune mechanisms regulating the development of different populations of FVIII-specific antibodies. The aim of the study described in this manuscript was the development of training and qualification test procedures to enable local operators in different European and US clinical Hemophilia Treatment Centers (HTC) to produce reliable and valid data for antigen-induced gene expression signatures in PBMC obtained from small blood volumes. For this purpose, we used the model antigen Cytomegalovirus (CMV) phosphoprotein (pp) 65. We trained and qualified 39 local HTC operators from 15 clinical sites in Europe and the US, of whom 31 operators passed the qualification at first attempt, and eight operators passed at the second attempt.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: research priorities to transform the care of people with hemophilia.

BACKGROUND: Decades of research have transformed hemophilia from severely limiting children's lives to a manageable disorder compatible with a full, active life, for many in high-income countries. The direction of future research will determine whether exciting developments truly advance health equity for all people with hemophilia (PWH). National Hemophilia Foundation (NHF) and American Thrombosis and Hemostasis Network conducted extensive inclusive all-stakeholder consultations to identify the priorities of people with inherited bleeding disorders and those who care for them. RESEARCH DESIGN AND METHODS: Working group (WG) 1 of the NHF State of the Science Research Summit distilled the community-identified priorities for hemophilia A and B into concrete research questions and scored their feasibility, impact, and risk. RESULTS: WG1 defined 63 top priority research questions concerning arthropathy/pain/bone health, inhibitors, diagnostics, gene therapy, the pediatric to adult transition of care, disparities faced by the community, and cardiovascular disease. This research has the potential to empower PWH to thrive despite lifelong comorbidities and achieve new standards of wellbeing, including psychosocial. CONCLUSIONS: Collaborative research and care delivery will be key to capitalizing on current and horizon treatments and harnessing technical advances to improve diagnostics and testing, to advance health equity for all PWH.

Hemophilia is the best known of the inherited bleeding disorders (BD). This is a rare condition that causes disproportionate bleeding, often into joints and vital organs. Factor replacement, injecting recombinant or plasma-based clotting factor products directly into the vein, became commonplace to control the disorder in the 1990s and 2000s. Prophylaxis, or injecting replacement factor every few days into people with hemophilia (PWH), has revolutionized patients' lives. In the last few years, other advances in new therapies have entered this space, such as non-factor replacement therapies and gene therapy. With many more research advances on the horizon, the National Hemophilia Foundation (NHF) initiated a State of the Science Research Summit in 2020. This event was attended by over 880 interested parties to help design an agenda of research priorities for inherited BDs for the next decade, based on community consultations. NHF formed multiple Working Groups (WG), each exploring a theme resulting from the community consultations, and presenting their results at the Summit. Led by 2 hematologists who manage and treat PWH daily, the 21-community member WG1 assigned to hemophilia A and B divided into 7 subgroups to identify and organize research priorities for different topic areas. The outcomes focused on prioritizing patients' needs, technological advances, and research in the areas of greatest potential for PWH and those who care for them. The results are a roadmap for the future execution of a research plan that truly serves the community.

Engineering a Therapeutic Protein to Enhance the Study of Anti-Drug Immunity.

The development of anti-drug antibodies represents a significant barrier to the utilization of protein-based therapies for a wide variety of diseases. While the rate of antibody formation can vary depending on the therapeutic employed and the target patient population receiving the drug, the antigen-specific immune response underlying the development of anti-drug antibodies often remains difficult to define. This is especially true for patients with hemophilia A who, following exposure, develop antibodies against the coagulation factor, factor VIII (FVIII). Models capable of studying this response in an antigen-specific manner have been lacking. To overcome this challenge, we engineered FVIII to contain a peptide (323-339) from the model antigen ovalbumin (OVA), a very common tool used to study antigen-specific immunity. FVIII with an OVA peptide (FVIII-OVA) retained clotting activity and possessed the ability to activate CD4 T cells specific to OVA323-339 in vitro. When compared to FVIII alone, FVIII-OVA also exhibited a similar level of immunogenicity, suggesting that the presence of OVA323-339 does not substantially alter the anti-FVIII immune response. Intriguingly, while little CD4 T cell response could be observed following exposure to FVIII-OVA alone, inclusion of anti-FVIII antibodies, recently shown to favorably modulate anti-FVIII immune responses, significantly enhanced CD4 T cell activation following FVIII-OVA exposure. These results demonstrate that model antigens can be incorporated into a therapeutic protein to study antigen-specific responses and more specifically that the CD4 T cell response to FVIII-OVA can be augmented by pre-existing anti-FVIII antibodies.

Recombinant porcine factor VIII corrects thrombin generation in vitro in plasma from patients with congenital hemophilia A and inhibitors.

Background: Neutralizing factor VIII (FVIII) antibodies are a major complication in hemophilia A. Antihemophilic factor VIII (recombinant), porcine sequence (rpFVIII; susoctocog alfa; Baxalta US Inc., a Takeda company) has low cross-reactivity to anti-human FVIII antibodies and can provide functional FVIII activity in the presence of FVIII inhibitors. Objectives: Evaluate in vitro thrombin generation and clot formation responses to rpFVIII in blood from patients with congenital hemophilia A. Methods: In this multicenter study, blood was obtained for in vitro analyses that included human and porcine FVIII inhibitors, low <5 Bethesda units (BU)/ml or high ≥5 BU/ml titer (Nijmegen-modified Bethesda assay); thrombin generation assay (TGA), clot viscoelasticity (thromboelastography), fibrin clot structure analysis (scanning electron microscopy), and epitope mapping. Results: Blood samples were from 20 patients with congenital hemophilia A (FVIII activity <1%, mean [range] inhibitor titers: anti-human FVIII, 14 [1-427] BU/ml [n = 13 high, n = 6 low, n = 1 data unavailable]); anti-porcine FVIII, 12 (0-886) BU/ml (n = 11 high, n = 8 low, n = 1 data unavailable). Porcine inhibitor titer and TGA response measured by endogenous thrombin potential showed an inverse correlation (2.7-10.8 U/ml rpFVIII Spearman correlation coefficient: -0.594 to -0.773; p < 0.01). Clot structures in low anti-porcine inhibitor titer plasmas were similar to those in noninhibitor plasma. Conclusions: Recombinant porcine factor VIII demonstrated a dose-dependent correction of thrombin generation and clot formation in vitro, dependent on the anti-porcine FVIII inhibitor titer. Procoagulant responses to rpFVIII occurred in plasma containing FVIII inhibitors.

Neutralizing Antibodies Against Factor VIII Can Occur Through a Non-Germinal Center Pathway.

Humoral immunity to factor VIII (FVIII) represents a significant challenge for the treatment of patients with hemophilia A. Current paradigms indicate that neutralizing antibodies against FVIII (inhibitors) occur through a classical CD4 T cell, germinal center (GC) dependent process. However, clinical observations suggest that the nature of the immune response to FVIII may differ between patients. While some patients produce persistent low or high inhibitor titers, others generate a transient response. Moreover, FVIII reactive memory B cells are only detectable in some patients with sustained inhibitor titers. The determinants regulating the type of immune response a patient develops, let alone how the immune response differs in these patients remains incompletely understood. One hypothesis is that polymorphisms within immunoregulatory genes alter the underlying immune response to FVIII, and thereby the inhibitor response. Consistent with this, studies report that inhibitor titers to FVIII differ in animals with the same F8 pathogenic variant but completely distinct backgrounds; though, how these genetic disparities affect the immune response to FVIII remains to be investigated. Given this, we sought to mechanistically dissect how genetics impact the underlying immune response to FVIII. In particular, as the risk of producing inhibitors is weakly associated with differences in HLA, we hypothesized that genetic factors other than HLA influence the immune response to FVIII and downstream inhibitor formation. Our data demonstrate that FVIII deficient mice encoding the same MHC and F8 variant produce disparate inhibitor titers, and that the type of inhibitor response formed associates with the ability to generate GCs. Interestingly, the formation of antibodies through a GC or non-GC pathway does not appear to be due to differences in CD4 T cell immunity, as the CD4 T cell response to an immunodominant epitope in FVIII was similar in these mice. These results indicate that genetics can impact the process by which inhibitors develop and may in part explain the apparent propensity of patients to form distinct inhibitor responses. Moreover, these data highlight an underappreciated immunological pathway of humoral immunity to FVIII and lay the groundwork for identification of biomarkers for the development of approaches to tolerize against FVIII.

Inhibitors: Diagnostic challenges, unknowns of inhibitor development, treatment of bleeding and surgery, and insights into diagnosis and treatment in China.

Factor (F) VIII inhibitors develop in around 30% of previously untreated patients (PUPs) with severe haemophilia, to a lesser extend in moderate and mild haemophilia A and in up to 10% in severe haemophilia B. Diagnostic challenges and questions remain including access to high quality testing, the role for functional inhibitor testing and binding antibody testing, and the adaptations needed in the presence of non-factor replacement therapy. Despite significant gains in knowledge there are still many unanswered questions underlying the immunologic mechanisms of inhibitor development and tolerance. Therapeutic options include eradication of inhibitors using immune tolerance induction therapy (ITI), prophylaxis with bypassing agents (i.e., recombinant activated factor VII /rFVIIa or activated prothrombin complex concentrate/aPCC) or non-factor replacement therapies (e.g., emicizumab) and treatment of bleeds or coverage of surgeries/invasive procedure. Recently a haemophilia centre capacity building program was launched in China to further develop the infrastructure and support needed to improve the diagnosis of haemophilia, detection of inhibitors, and continue to improve the care of patients with haemophilia and inhibitors.

Eptacog beta efficacy and safety in the treatment and control of bleeding in paediatric subjects (<12 years) with haemophilia A or B with inhibitors.

INTRODUCTION: Eptacog beta is a new recombinant activated human factor VII bypassing agent approved in the United States for the treatment and control of bleeding in patients with haemophilia A or B with inhibitors 12 years of age or older. AIM: To prospectively assess in a phase 3 clinical trial (PERSEPT 2) eptacog beta efficacy and safety for treatment of bleeding in children <12 years of age with haemophilia A or B with inhibitors. METHODS: Using a randomised crossover design, subjects received initial doses of 75 or 225 µg/kg eptacog beta followed by 75 µg/kg dosing at predefined intervals (as determined by clinical response) to treat bleeding episodes (BEs). Treatment success criteria included a haemostasis evaluation of 'excellent' or 'good' without use of additional eptacog beta, alternative haemostatic agent or blood product, and no increase in pain following the first 'excellent' or 'good' assessment. RESULTS: Treatment success proportions in 25 subjects (1-11 years) who experienced 546 mild or moderate BEs were 65% in the 75 µg/kg initial dose regimen (IDR) and 60% in the 225 µg/kg IDR 12 h following initial eptacog beta infusion. By 24 h, the treatment success proportions were 97% for the 75 µg/kg IDR and 98% for the 225 µg/kg IDR. No thrombotic events, allergic reactions, neutralising antibodies or treatment-related adverse events were reported. CONCLUSION: Both 75 and 225 µg/kg eptacog beta IDRs provided safe and effective treatment and control of bleeding in children <12 years of age.

Nonhuman glycans can regulate anti-factor VIII antibody formation in mice.

Recombinant factor VIII (FVIII) products represent a life-saving intervention for patients with hemophilia A. However, patients can develop antibodies against FVIII that prevent its function and directly increase morbidity and mortality. The development of anti-FVIII antibodies varies depending on the type of recombinant product used, with previous studies suggesting that second-generation baby hamster kidney (BHK)-derived FVIII products display greater immunogenicity than do third-generation Chinese hamster ovary (CHO)-derived FVIII products. However, the underlying mechanisms responsible for these differences remain incompletely understood. Our results demonstrate that BHK cells express higher levels of the nonhuman carbohydrate α1-3 galactose (αGal) than do CHO cells, suggesting that αGal incorporation onto FVIII may result in anti-αGal antibody recognition that could positively influence the development of anti-FVIII antibodies. Consistent with this, BHK-derived FVIII exhibits increased levels of αGal, which corresponds to increased reactivity with anti-αGal antibodies. Infusion of BHK-derived, but not CHO-derived, FVIII into αGal-knockout mice, which spontaneously generate anti-αGal antibodies, results in significantly higher anti-FVIII antibody formation, suggesting that the increased levels of αGal on BHK-derived FVIII can influence immunogenicity. These results suggest that posttranslational modifications of recombinant FVIII products with nonhuman carbohydrates may influence the development of anti-FVIII antibodies.

Successful Perioperative Management of Orthotopic Cardiac Transplantation in a Pediatric Patient With Concurrent Congenital von Willebrand Disease and Acquired von Willebrand Syndrome Using Recombinant von Willebrand Factor.

Von Willebrand disease (VWD) is the most common bleeding disorder and reportedly affects 1:1,000 of the world's population. There are three subtypes of VWD characterized by a quantitative defect (types 1 and 3 VWD) or a qualitative defect (type 2 VWD). Type 1 VWD results in a partial deficiency of von Willebrand factor (VWF) and affects approximately 75% of individuals with VWD, whereas type 3 VWD results in a severe or complete deficiency of VWF. Individuals with type 2 VWD subtypes (types 2A, 2B, 2M, and 2N VWD) express a dysfunctional VWF protein that has impaired interactions with platelets or factor VIII. The majority of individuals with VWD have mild type 1 VWD and occasionally require bolus infusions of VWF for severe bleeding or major surgery. A subset of patients, especially those with type 2A or 3 VWD, may require more frequent VWF replacement or prophylaxis for refractory bleeding or bleeding prevention, respectively. Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder that primarily occurs as a result of an underlying disease or other pathologic mechanism. Cases of AVWS associated with heart valve defects, left ventricular assist devices, or congenital cardiac disease result from high shear stress in the circulation that induces VWF unfolding and subsequent proteolysis of high-molecular-weight multimers by ADAMTS-13. In rare instances, plasma-derived factor VIII-containing VWF concentrates have been administered to individuals with AVWS for persistent or challenging bleeding events. In this case report, the hemostatic challenges and the perioperative management of cardiac transplantation surgery using a novel recombinant VWF product in a pediatric patient diagnosed with AVWS concomitant with congenital type 1 VWD are described. Written informed consent was obtained from the patient's mother for this case report. The diagnosis of congenital VWD remains a challenge because of multiple potential modifiers that can alter VWF laboratory results. Concurrent conditions, such as congenital heart disease and the rare secondary condition of AVWS, in addition to congenital VWD, can further affect interpretation of coagulation studies. This can result in delays in diagnosis, increase severity of the bleeding phenotype, and complicate hemostatic management in individuals at risk for bleeding and thrombosis. A multidisciplinary approach, including anesthesiologists, cardiologists, cardiovascular surgeons, hematologists, and pharmacists, is critical to achieving optimal patient outcomes, as highlighted in this case report. As diagnostic capabilities and understanding of VWD broaden, future studies evaluating alternative treatment approaches for individuals with various types of VWD would be of great benefit to the medical community.

Emicizumab in tolerized patients with hemophilia A with inhibitors: A single-institution pediatric cohort assessing inhibitor status.

BACKGROUND: The majority of patients with hemophilia A with inhibitors who undergo immune tolerance induction (ITI) achieve successful tolerance and transition to factor VIII (FVIII) prophylaxis. A portion of these patients have switched to emicizumab for bleeding prevention. However, the risk of inhibitor relapse on emicizumab is unclear. OBJECTIVE: To evaluate the inhibitor status of patients with hemophilia A and inhibitors who achieved successful/partial tolerance after ITI and transitioned from FVIII prophylaxis to emicizumab. METHODS: This is a single-institution, retrospective review of pediatric patients with severe hemophilia A who have completed ITI with FVIII and switched to emicizumab. RESULTS/CONCLUSIONS: Seven successfully tolerized and five partially tolerized patients were identified. Three patients continued intermittent FVIII infusions on emicizumab at 50-70 IU/kg twice weekly, once weekly, or every other week due to concerns for inhibitor relapse or loss of recent FVIII tolerance by the treating provider. Eleven of 12 patients (92%) maintained negative inhibitor titers at a mean follow-up of 14.2 ± 6.1 months. One individual had an inhibitor relapse with a peak titer of 2.5 BU/mL. Five of the 11 patients (45%) with negative inhibitor titers had detectable nonneutralizing anti-FVIII IgG4 antibodies, but none of the patients had detectable IgG1 antibodies. There were no inhibitor recurrences in a subset of six patients after FVIII re-exposure for bleeding events or surgery. Given that the presence of an inhibitor significantly impacts factor product choice for bleeding management, ongoing inhibitor monitoring in tolerized patients with hemophilia A who transition to emicizumab is strongly recommended.

B cell-activating factor modulates the factor VIII immune response in hemophilia A.

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell-activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody-mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.

Emerging benefits of Fc fusion technology in the context of recombinant factor VIII replacement therapy.

Although the primary reason for recombinant factor VIII Fc fusion protein (rFVIIIFc) development was to reduce treatment burden associated with routine prophylaxis, new evidence suggests additional benefits of Fc fusion technology in the treatment of people with haemophilia A. Preclinical research has been utilized to characterize the potential immunomodulatory properties of rFVIIIFc, including an ability to reduce inflammation and induce tolerance to factor VIII. This has since been expanded into clinical research in immune tolerance induction (ITI) with rFVIIIFc, results of which suggest the potential for rapid tolerization in first-time ITI patients and therapeutic benefit in patients undergoing rescue ITI. The potential for improved joint health through the anti-inflammatory properties of rFVIIIFc has also been suggested. In addition, a new avenue of research into the role of rFVIIIFc in promoting bone health in patients with haemophilia A, potentially through reduced osteoclast formation, has yielded encouraging results that support further study. This review summarizes the existing preclinical and clinical studies of immunomodulation and tolerization with rFVIIIFc, as well as studies in joint and bone health, to elucidate the potential benefits of rFVIIIFc in haemophilia A beyond the extension of factor VIII half-life.

Fc Gamma Receptors and Complement Component 3 Facilitate Anti-fVIII Antibody Formation.

Anti-factor VIII (fVIII) alloantibodies, which can develop in patients with hemophilia A, limit the therapeutic options and increase morbidity and mortality of these patients. However, the factors that influence anti-fVIII antibody development remain incompletely understood. Recent studies suggest that Fc gamma receptors (FcγRs) may facilitate recognition and uptake of fVIII by recently developed or pre-existing naturally occurring anti-fVIII antibodies, providing a mechanism whereby the immune system may recognize fVIII following infusion. However, the role of FcγRs in anti-fVIII antibody formation remains unknown. In order to define the influence of FcγRs on the development of anti-fVIII antibodies, fVIII was injected into WT or FcγR knockout recipients, followed by evaluation of anti-fVIII antibodies. Anti-fVIII antibodies were readily observed following fVIII injection into FcγR knockouts, with similar anti-fVIII antibody levels occurring in FcγR knockouts as detected in WT mice injected in parallel. As antibodies can also fix complement, providing a potential mechanism whereby anti-fVIII antibodies may influence anti-fVIII antibody formation independent of FcγRs, fVIII was also injected into complement component 3 (C3) knockout recipients in parallel. Similar to FcγR knockouts, C3 knockout recipients developed a robust response to fVIII, which was likewise similar to that observed in WT recipients. As FcγRs or C3 may compensate for each other in recipients only deficient in FcγRs or C3 alone, we generated mice deficient in both FcγRs and C3 to test for potential antibody effector redundancy in anti-fVIII antibody formation. Infusion of fVIII into FcγRs and C3 (FcγR × C3) double knockouts likewise induced anti-fVIII antibodies. However, unlike individual knockouts, anti-fVIII antibodies in FcγRs × C3 knockouts were initially lower than WT recipients, although anti-fVIII antibodies increased to WT levels following additional fVIII exposure. In contrast, infusion of RBCs expressing distinct alloantigens into FcγRs, C3 or FcγR × C3 knockout recipients either failed to change anti-RBC levels when compared to WT recipients or actually increased antibody responses, depending on the target antigen. Taken together, these results suggest FcγRs and C3 can differentially impact antibody formation following exposure to distinct alloantigens and that FcγRs and C3 work in concert to facilitate early anti-fVIII antibody formation.

Discordance between platelet-supported and vesicle-supported factor VIII activity in the presence of anti-C2 domain inhibitory antibodies.

BACKGROUND: We recently reported that factor VIII (FVIII) binds to a macromolecular complex including fibrin on thrombin-stimulated platelets and that two antibodies against FVIII diminish platelet-supported FVIII activity more than vesicle-supported activity. The C2 domain of FVIII is known to bind to phospholipid membrane and also binds fibrin. OBJECTIVES: We asked whether the degree of inhibition by anti-C2 antibodies would show differences between platelet-supported and the standard activated partial thromboplastin time (aPTT) assay. METHODS: We evaluated the inhibition by a well-defined panel of monoclonal anti-C2 domain antibodies encompassing the major epitopes of the C2 domain. Activity was measured in an activated platelet time (aPT) assay containing fresh, density gradient-purified human platelets. RESULTS: The aPT exhibited a log-linear relationship between FVIII and time to fibrin formation over a 4-log range, encompassing 0.01% to 100% plasma FVIII. Nine of 10 mAbs inhibited 89% to 96% of FVIII activity, whereas mAb F85 did not. There was no correlation between the degree of inhibition in the aPTT-based assay and the platelet assay. In particular, four mAbs did not inhibit the aPTT assay, yet inhibited 90% of platelet-based activity. Residual FVIII activity in purified-protein assays, relying on platelets, correlated with the aPT assay. CONCLUSIONS: The degree of FVIII impairment by some inhibitor antibodies is substantially different on platelet membranes vs synthetic vesicles. Thus, current inhibitor assays may underestimate the frequency of significant inhibitors, and a platelet-based assay may more accurately assess bleeding risk.

Development and application of global assays of hyper- and hypofibrinolysis.

Numerous methods for evaluation of global fibrinolytic activity in whole blood or plasma have been proposed, with the majority based on tissue-type plasminogen activator (t-PA) addition to initiate fibrinolysis. We propose that such an approach is useful to reveal hypofibrinolysis, but t-PA concentrations should be kept to a minimum. In this paper, we describe a low-concentration t-PA plasma turbidity assay to evaluate several congenital factor deficiencies, including plasminogen activator inhibitor-1 (PAI-1) and plasminogen deficiency, as well as hemophilia A and B. In addition, we demonstrate a threshold dependency on endogenous PAI-1 levels. To assess endogenous hyperfibrinolysis, we suggest that assays that avoid t-PA addition are preferable, with assays based on euglobulin fractionation remaining a viable choice. We describe a euglobulin fraction clot lysis time (ECLT) assay with spectrophotometric readout and other modifications, and evaluate it as a tool to measure hyperfibrinolysis in inherited clotting factor deficiency states. We demonstrate that the ECLT is predominantly driven by residual amounts of PAI-1, t-PA, and α2-antiplasmin. These assays should be further evaluated for the detection of hypo- or hyperfibrinolysis in acquired thrombotic or hemorrhagic disorders.

The evolution of factor VIIa in the treatment of bleeding in haemophilia with inhibitors.

The use of activated factor VII (FVIIa) for the treatment of bleeding events in haemophilia patients with inhibitors was first reported over 30 years ago. Since then clinical trials, registries, case series, real-world experience and an understanding of its mechanism of action have transformed what was originally a scientific curiosity into one of the major treatments for inhibitor patients, with innovative therapeutic regimens, dose optimization and individualized care now widely practiced. Given current understanding and use, it might be easy to forget the years of clinical research that led up to this point; in this review, we lay out changes based on broad eras of rFVIIa use. These eras cover the original uncertainty associated with dosing, efficacy and safety; the transformation of care ushered in with its widespread use; and the optimization and individualization of patient care and the importance of specialized support provided by haemophilia treatment centres. Today with the introduction of novel prophylactic agents such as emicizumab, we once again find ourselves dealing with the uncertainties of how best to utilize rFVIIa and newer investigational variants such as marzeptacog alfa and eptacog beta; we hope that the experiences of the past three decades will serve as a guide for this new era of care.

Immune tolerance induction in paediatric patients with haemophilia A and inhibitors receiving emicizumab prophylaxis.

INTRODUCTION: The formation of neutralizing antifactor VIII (fVIII) antibodies, called inhibitors, is the most common complication in modern haemophilia A care. Novel non-factor replacement therapies, such as emicizumab, have sought to address the limitations of bypassing agents for bleeding management in patients with inhibitors. However, immune tolerance induction (ITI) remains the primary method for eradicating inhibitors and restoring the hemostatic response to fVIII. AIM: The aim of this study was to review a case series of paediatric patients with haemophilia A and inhibitors who have received ITI for inhibitor eradication concomitantly with emicizumab prophylaxis for bleeding prevention. METHODS: Single institution retrospective chart review of paediatric patients with severe haemophilia A receiving ITI and emicizumab. RESULTS: Seven patients were included in this cohort. Six patients used three different recombinant fVIII products at 100 IU/kg three times per week, and one patient used a plasma-derived fVIII product at an initial dose of 50 IU/kg three times per week. Three patients achieved a negative inhibitor titre <0.6 Chromogenic Bethesda Units per mL (CBU/mL), and two patients achieved a normal fVIII recovery ≥66%. There were nine bleeding events in four patients, but no thrombotic events. All patients remained on ITI and emicizumab. CONCLUSION: Immune tolerance induction while on emicizumab prophylaxis is a feasible approach in paediatric haemophilia A patients with inhibitors. This is the first report of the concomitant use of ITI in patients receiving emicizumab prophylaxis described as the 'Atlanta Protocol'.