anti-FVIII Antibodies Research Articles

Concizumab improves clot formation in hemophilia A under flow

BackgroundInhibition of tissue factor pathway inhibitor (TFPI) is an emerging therapeutic strategy for treatment of hemophilia. Concizumab is a monoclonal antibody that binds TFPI and blocks its inhibition of factor (F)Xa thereby extending the initiation of coagulation and compensating for lack of FVIII or FIX. ObjectivesThe objective of this in vitro study was to evaluate how concizumab affects clot formation in hemophilia A under flow. MethodsBlood was collected from normal controls or people with hemophilia A. An anti-FVIII antibody was added to normal controls to simulate hemophilia A with inhibitory antibodies to FVIII. Whole blood and recombinant activated FVII (rFVIIa, 25 nM) or concizumab (200, 1000, and 4000 ng/mL) were perfused at 100 s−1 over a surface micropatterned with tissue factor (TF) and collagen-related peptide. Platelet and fibrin(ogen) accumulation were measured by confocal microscopy. Static thrombin generation in plasma was measured in response to rFVIIa and concizumab. ResultsConcizumab (1000 and 4000 ng/mL) and rFVIIa both rescued (93%-101%) total platelet accumulation, but only partially rescued (53%-63%) fibrin(ogen) incorporation to normal control levels in simulated hemophilia A. Results using congenital hemophilia A blood confirmed effects of rFVIIa and concizumab. While these 2 agents had similar effect on clot formation under flow, concizumab enhanced thrombin generation in plasma under static conditions to a greater extent than rFVIIa. ConclusionTFPI inhibition by concizumab enhanced activation and aggregation of platelets and fibrin clot formation in hemophilia A to levels comparable with that of rFVIIa.

The effects of emicizumab on in vitro coagulation and fibrinolysis parameters in patients with disseminated intravascular coagulation with and without addition of anti-FVIII antibody.

Emicizumab (Emi) is used as haemostatic prophylaxis for patients with haemophilia A (PwHA). Disseminated intravascular coagulation (DIC) is a condition characterized by persistent systemic activation of coagulation, but there is yet no information on coagulation and fibrinolysis potentials in Emi-treated PwHA with DIC. To examine the effect of Emi on coagulation and fibrinolysis potentials in HA-model DIC plasmas. Plasma from a patient with sepsis-DIC (seven patients) was treated with anti-factor (F)VIII monoclonal antibody (HA-model DIC plasma) and incubated with Emi (50µg/mL). The plasma was then assessed using clot-fibrinolysis waveform analysis (CFWA). Coagulation and fibrinolysis parameters were expressed as ratios relative to normal plasma (|min1|-ratio and |FL-min1|-ratio, respectively). In case 1, coagulant potential was slightly high and fibrinolytic potential was extremely low, presenting a coagulant-dominant state (|min1|-ratio/|FL-min1|-ratio: 1.1/.38). In cases 2-5, fibrinolytic potential was not suppressed, but there were marked hypercoagulant potentials, indicating relative coagulant-dominant states. In case 6, coagulant and fibrinolytic potentials were increased but well balanced (|min1|-ratio/|FL-min1|-ratio: 1.38/1.28). In case 7, both potentials were severely deteriorated in not only CFWA but also the thrombin/plasmin generation assay. The addition of Emi into the HA-model DIC plasmas increased |min1|-ratio values in all cases, but the coagulant potentials did not exceed the initial ones (DIC plasma before treatment with anti-FVIII antibody). The presence of Emi in the HA-model DIC plasma improved coagulation potentials, but did not increase coagulation potentials beyond those of DIC plasma in non-HA states.

Comprehensive domain-specific analysis and immunoglobulin G profiling of anti–factor VIII antibodies using a bead-based multiplex immunoassay

BackgroundAntibodies against factor (F)VIII are a major complication in the treatment of patients with severe hemophilia A. The Nijmegen-Bethesda assay (NBA) is the gold standard for detection of neutralizing antibodies (inhibitors), whereas both inhibitors and nonneutralizing antibodies can be detected by immunoassays such as enzyme-linked immunosorbent assay (ELISA) and multiplex bead-based assays. ObjectivesEvaluation of an in-house Luminex bead-based assay (LumiTope) compared with a commercially available ELISA and NBA. MethodsThe LumiTope method comprised full-length and B-domain–deleted FVIII as well as 9 purified FVIII single or multidomains. The respective proteins were coupled to magnetic beads to detect domain-specific immunoglobulin (IgG; IgG1-4) anti-FVIII antibodies in a large cohort of patients with hemophilia A with and without inhibitors. ResultsOverall, LumiTope assay had a high sensitivity (94.9%) and specificity (91.2%), particularly in patients with low-titer inhibitors compared with ELISA (sensitivity of 72.2% vs 27.7%). IgG4 was the most abundant IgG subclass in NBA-positive patients. NBA-positive and -negative patients showed different domain profiles. Patients with genetic variants in the heavy chain predominantly exhibited antibodies specific to this chain, while those with a light-chain variant showed a more diverse distribution of antibody specificities. Patients with an intron 22 inversion resembled those with a light-chain defect, with a majority of antibodies targeting the light chain. ConclusionLumiTope assay provides a sensitive and specific method for not only detection but also domain specification of anti-FVIII-antibodies. Implementation of bead-based assays could improve antibody detection, profiling, and comparability of results and complement NBA.

Study of Factor VIII Inhibitor and F8 Gene Mutations in Persons with Hemophilia A from Benin

Aim: determine the prevalence of a FVIII inhibitors and identify the genetic mutations associated with their development in beninese personne with hemophilia (PwH) A. Study Design: this is cross-sectional descrptive study conducted from June 2022 to May 2023 in hemophilia treatment centers in Benin. Methodology: Inhibitor screening was carried out systematically in all PwHs A receiving FVIII infusion through determination of the circulating anticoagulant index and the Nijmegen-Bethesda assay. The molecular study strategy used for the F8 gene associated with hemophilia A is dependent on the severity of the hemophilia. Other data were collected either from patients' responses to the questionnaire or by studying their medical records and the center's hemophilia registry. Results: Of the 97 PwHs A followed up, 57 had been treated with FVIII infusion. Of these, 21 had developed inhibitors, representing a frequency of 36.8% of treated PwHs A and 43.75% of severe PwHs. None of the moderate or mild PwHs A had developed anti-FVIII antibodies. PwHs A with inhibitors had a median age of 11 years, ranging from 1 to 66 years. The Nijmegen-Bethesda test revealed 11 high responders and 10 low responders. Mutation analysis of the F8 gene revealed seven cases of intron 22 inversion, seven cases of nonsense mutations, three cases of deletion and one case of missense mutation. Mutations weren’t identified in three patients because their DNA did not amplify on long-distance PCR. In terms of therapy, immune tolerance induction wasn’t achieved in any of the 21 patients, but they are treated with emicizumab and bypass depending on the context. Conclusion: Although a cross-sectional study with a limited sample size, this study provides valuable information on beninese PwHs A with inhibitors. The frequency of inhibitors is high in treated PwHs A, and almost all patients who have developed inhibitors have high-risk genetic mutations.

The spectrum of neutralizing and non-neutralizing anti-FVIII antibodies in a nationwide cohort of 788 persons with hemophilia A.

Anti-factor VIII (FVIII) antibodies have been reported to exhibit both neutralizing and non-neutralizing characteristics. This is the first study investigating the full spectrum of FVIII-specific antibodies, including non-neutralizing antibodies, very-low titer inhibitors, and inhibitors, in a large nationwide population of persons with hemophilia A of all severities. All persons with hemophilia A (mild (FVIII > 5-40 IU/dL)/moderate [FVIII 1-5 IU/dL)/severe (FVIII < 1 IU/dL)] with an available plasma sample who participated in the sixth Hemophilia in the Netherlands study between 2018 and 2019 were included. The presence of anti-FVIII antibodies of the immunoglobulin A, M, and G isotypes and IgG subclasses, along with antibody titer levels, were assessed using direct-binding ELISAs. FVIII specificity was assessed using a competition-based ELISA approach. The inhibitor status was determined using the Nijmegen ultra-sensitive Bethesda assay (NusBA) and the Nijmegen Bethesda assay (NBA). In total, 788 persons with hemophilia A (336 (42.6%) mild, 123 (15.6%) moderate, 329 (41.8%) severe hemophilia) were included. The median age was 45 years (IQR 24-60), and the majority (50.9%) had over 150 exposure days to FVIII concentrates. Within our population, 144 (18.3%) individuals had non-neutralizing FVIII-specific antibodies, 10 (1.3%) had very low-titer inhibitors (NusBA positive; NBA negative), and 13 (1.6%) had inhibitors (both NusBA and NBA positive). IgG1 was the most abundant FVIII-specific antibody subclass, and the highest titer levels were found for IgG4. In individuals without a reported history of inhibitor development, no clear differences were observed in antibody patterns between those who were minimally or highly exposed to FVIII concentrates. IgG4 subclass antibodies were only observed in persons with a reported history of FVIII inhibitor or in those with a currently detected (very low-titer) inhibitor. In this cross-sectional study, we identified non-neutralizing antibodies in a relatively large proportion of persons with hemophilia A. In contrast, in our population, consisting of persons highly exposed to FVIII concentrates, (very low-titer) inhibitors were detected only in a small proportion of persons, reflecting a well-tolerized population. Hence, our findings suggest that only a small subpopulation of non-neutralizing FVIII-specific antibodies is associated with clinically relevant inhibitors.

Immune tolerance promotion by LSEC-specific lentiviral vector-mediated expression of the transgene regulated by the stabilin-2 promoter

Liver sinusoidal endothelial cells (LSECs) are specialized endocytic cells that clear the body from blood-borne pathogens and waste macromolecules through scavenger receptors (SRs). Among the various SRs expressed by LSECs is stabilin-2 (STAB2), a class H SR that binds to several ligands, among which endogenous coagulation products. Given the well-established tolerogenic function of LSECs, we asked whether the STAB2 promoter (STAB2p) would enable us to achieve LSEC-specific lentiviral vector (LV)-mediated transgene expression, and whether the expression of this transgene would be maintained over the long-term due to tolerance induction. Here we show that STAB2p ensures LSEC-specific green fluorescent protein (GFP) expression by LV in the absence of a specific cytotoxic CD8+ T cell immune response, even in the presence of GFP-specific CD8+ T cells, confirming the robust tolerogenic function of LSECs. Finally, we show that our delivery system can partially and permanently restore FVIII activity in a mouse model of severe hemophilia A without the formation of anti-FVIII antibodies. Overall, our findings establish the suitability of STAB2p for long-term LSEC-restricted expression of therapeutic proteins, such as FVIII, or to achieve antigen-specific immune tolerance in auto-immune diseases.

Simoctocog alfa (Nuwiq®) in children: early steps in life's journey for people with severe hemophilia A.

People with severe hemophilia A usually experience their first bleed early in life. In children with severe hemophilia A, primary prophylaxis is recommended to prevent recurrent and potentially life-threatening bleeds that significantly impact day-to-day life. Factor VIII (FVIII) prophylaxis is well-established in children and has been shown to reduce the development of hemophilic arthropathy. However, a major challenge of FVIII therapy is the development of neutralizing anti-FVIII antibodies (FVIII inhibitors). Simoctocog alfa (Nuwiq®) is a human cell line-derived recombinant FVIII (rFVIII) whose immunogenicity, efficacy, and safety have been studied in 167 children with severe hemophilia A across two prospective clinical trials and their long-term extensions. In 105 previously untreated children, the inhibitor rate of 16.2% for high-titer inhibitors (26.7% for all inhibitors) was lower than published rates for hamster cell line-derived rFVIII products. There was no inhibitor development in previously untreated children with non-null F8 mutations and in previously treated children. In a case series of 10 inhibitor patients, 8 (80%) underwent successful immune tolerance induction with simoctocog alfa with a median time to undetectable inhibitor of 3.5 months. In an analysis of 96 children who enrolled in the extension studies and received long-term simoctocog alfa prophylaxis for up to 5 years, median spontaneous, joint, and total annualized bleeding rates were 0.3, 0.4, and 1.8, respectively. No thromboembolisms were reported in any of the 167 children, and there were no treatment-related deaths. Optimal care of children should consider several factors, including minimization of inhibitor development risk, maintaining tolerance to FVIII, highly effective bleed prevention and treatment, safety, and impact on long-term outcomes such as bone and joint health. In this context we review the pediatric clinical data and ongoing studies with simoctocog alfa.

Emicizumab-mediated hemostatic function assessed by thrombin generation assay in an in vitro model of factor VIII-depleted thrombophilia plasma.

Patients with hemophilia A (PwHA) may have concurrent deficiency of representative anticoagulant proteins, protein (P)C, PS, and antithrombin (AT), which reduces bleeding frequency. However, emicizumab-driven hemostasis in PwHA with such thrombophilic potential remains unclarified. This study investigated the influence of natural anticoagulants on emicizumab-driven coagulation in HA model plasma. Various concentrations of PS and AT were added to PS-deficient plasma and AT-deficient plasma in the presence of anti-FVIII antibody (FVIIIAb; 10BU/mL). PC-deficient plasma was mixed with normal plasma at various concentrations in the presence of FVIIIAb. Emicizumab (50µg/mL) was added to these thrombophilic HA model plasmas, prior to tissue factor/ellagic acid-triggered thrombin generation assays. Co-presence of emicizumab increased peak thrombin values (PeakTh) dependent on PS, AT, and PC concentrations. Maximum coagulation potentials in the PS-reduced HA model plasmas remained normal in the presence of emicizumab. PeakTh were close to normal in the presence of 50%AT irrespective of emicizumab, but were higher than normal in the presence of 25%AT. Addition of recombinant FVIIa (corresponding to an administered dose of 90μg/kg) enhanced coagulation potential to normal levels. Our findings provide novel information on hemostatic regulation in emicizumab-treated PwHA with a possible thrombophilic disposition.

Hybrid human-porcine factor VIII proteins partially escape the inhibitory effects of anti-factor VIII inhibitor alloantibodies having A2 or C2 domain specificity.

Porcine factor (pF)VIII has low cross-reactivity with anti-human (h)FVIII inhibitor alloantibodies. Clinical trials of pFVIII in congenital haemophilia A patients with inhibitor (PwHA-I) are in progress. Most polyclonal anti-hFVIII inhibitors recognize its A2 and/or C2 domain(s), and recombinant human-porcine hybrid (hp)FVIII proteins may escape neutralization by these inhibitors. To evaluate the ability of hpFVIII to limit the anti-FVIII activity of inhibitor alloantibodies. Three hybrid proteins were created by substituting the hFVIII A2, C2 domain or both with the corresponding domains of pFVIII [termed hp(A2), hp(C2) and hp(A2/C2), respectively]. The reactivity of these hybrids was assessed by one-stage clotting assays (OSA), thrombin generation assays (TGA) and rotational thromboelastometry (ROTEM) by adding them to FVIII-deficient samples. OSA demonstrated that the hybrid proteins avoided neutralization by anti-FVIII A2 or C2 monoclonal antibodies (mAb) and polyclonal inhibitor-antibodies (polyAb) from PwHA-I. In TGA, thrombin generation with hp(A2) and hp(A2/C2) was not attenuated in the presence of patient IgG recognizing anti-A2 domain. In contrast, that with hFVIII and hp(C2) was suppressed by this IgG to levels equivalent to those of FVIII-deficient plasma. With anti-A2/C2 polyAb, the activity of hp(A2/C2) was unaffected. ROTEM demonstrated that the addition of hp(A2) or hp(A2/C2) to anti-A2 polyAb shortened clot times/clot formation times, whilst hFVIII or hp(C2) were ineffective. Similarly with anti-A2/C2 polyAb, hp(A2/C2) restored coagulation potential to a greater extent than hp(A2) and hp(C2). Hybrid FVIII proteins containing porcine FVIII A2 and/or C2 domain(s) could support effective therapy in PwHA-I by avoiding neutralization.

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

Hemophilia A Research Program (HARP): A Novel Study Design for a Multicenter and Decentralized Prospective Observational Longitudinal Cohort Study in Hemophilia A

Background: Hemophilia A is a rare X-linked bleeding disorder caused by factor VIII (FVIII) deficiency. There is a compelling need in hemophilia to better understand the causes of bleeding in females and the factors underlying FVIII inhibitor development. To meet this need we are establishing a new NHLBI funded Hemophilia A Analytical Cohort Research Program (HARP) as part of a public-private partnership to support an Intergenerational Precision Medicine Program for the study of factor VIII (FVIII) immunogenicity in severe hemophilia A (Harpf8.org). The HARP objectives are: 1) Longitudinal assessment of bleeding in females with a severe hemophilia A genotype beginning in pregnancy; 2) Longitudinal assessment of anti-FVIII antibody formation in severe hemophilia A from birth through at least the first 2 years of life; 3) Studies of causes of bleeding in females with a severe hemophilia A genotype; 4) Studies of the immune response to FVIII in severe hemophilia A; 5) Development of a well-curated annotated data set of genetic, hemophilia, immune, environmental, psychosocial, and quality of life metrics; 6) Development of a multi-omics and immunologic biospecimen resource; and 7) Development of the hemophilia analytical Portal (F8portal.org). Study design: The HARP is a multicenter and decentralized prospective observational longitudinal cohort study beginning in pregnancy. It will follow 50 mother-baby pairs where the babies have severe hemophilia A and the mothers have a severe hemophilia A genotype. The private Clinical Consortium (CC) will oversee the study throughout the U.S., including sites that care for the mothers (up to 200 sites) and sites that care for the child (up to 70 sites). The CC will also conduct decentralized aspects of the study including collection of patient reported outcomes and local sample collection. The primary maternal endpoint is primary postpartum hemorrhage; the primary pediatric endpoint is rate of development of humoral immune response to FVIII and proportion that progress to clinical inhibitors. Mother and child secondary endpoints reflect the diverse research the HARP resource will support. Inclusion criteria for the pregnant person are: Pregnant and prior to 37 weeks gestation, known or at-risk to have a severe hemophilia A genotype, pregnant with a fetus at-risk for severe hemophilia A, ability to give informed consent, and &amp;gt; 18 years old. Inclusion criteria for the child are: severe hemophilia A, born to a person in the study. Exclusion/discontinuation criteria are: Presence of another significant bleeding disorder in mother or child; mother age &amp;lt;18 years; no fetus is at-risk to inherit severe hemophilia A; the child does not have severe hemophilia A; the child has a severe immune disorder; the mother or child participates in another study with blood collection volumes that exceed safety limits; mother's or child's healthcare will be outside the U.S.; the mother is a prisoner; any other reason that in the opinion of the investigator a subject is unsuitable for the study Summary: The HARP innovates in multiple areas of clinical study design to conduct a national prospective longitudinal intergenerational study in a rare disorder beginning in pregnancy. The structure includes a new multidisciplinary CC, systematic inclusion of Lived Experience Experts, strategies for broad community engagement, recruitment, and retention, and decentralized operations. This cohort will enable deep characterization, including genomics and other -omics, on pregnancy-associated bleeding and drivers of FVIII inhibitors in hemophilia A. The HARP Shareable Resource will be comprised of well annotated data and biological samples accessible to researchers through a portal, F8portal.org, and a linked biorepository.

Impact of Recombinant Factor VIII and Platelet Interaction on Platelet Functionality and Hemophilia a Treatment

Background: Platelets play an important role throughout the various stages of hemostasis. After vascular injury, platelets become activated in a pro-aggregatory state, and subsequently a subpopulation undergoes a phenotype shift to a pro-coagulant state. Pro-coagulant platelets bind to factor VIII (FVIII) and ensure efficient localization of FVIII at the site of injury. It is unclear whether modifications in recombinant FVIII (rFVIII) products impact FVIII-platelet binding and subsequent platelet signaling. Aims: To examine binding of different rFVIII products to platelets in vitro, and the impact of rFVIII on platelet phenotype and intracellular signaling. Methods: Platelet activation: Platelets isolated from healthy donors were activated with thrombin and cross-linked collagen-related peptide (CRP-XL). Platelet-FVIII binding: Activated platelets were incubated with simoctocog alfa, efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding was quantified by flow cytometry either by immunostaining with an anti-FVIII antibody conjugated to Alexa fluor (AF)647 or direct measurement of rFVIII concentrates labelled with AF647. Phenotype shift: During the shift to a pro-coagulant state, phosphatidylserine (PS) becomes exposed on the platelet membranes, with the extent of PS exposure correlating with the level of FVIII binding. To assess PS exposure, activated platelets were incubated with Annexin V-BV421, a molecular probe with high affinity for PS. Inhibition of integrin αIIbβ3 signaling: Activated platelets were incubated with the integrin αIIbβ3 inhibitor antibody 10E5. Results: Binding to pro-coagulant platelets was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol irrespective of immunostaining method (p&amp;lt;0.05; Figure 1 shows detection with anti-FVIII antibody conjugated with AF647).To validate the variability in rFVIII-platelet interactions observed, we performed a dynamic platelet-binding assay with the two FVIII products exhibiting the strongest binding (simoctocog alfa and efmoroctocog alfa). The results of the dynamic assay confirmed the results of the static experiments (Figure 1). Exposure of activated platelets to simoctocog alfa and efmoroctocog alfa resulted in increased PS exposure, with a greater effect observed with simoctocog alfa, consistent with the results of the binding experiments. Immunostaining experiments revealed co-localization of simoctocog alfa and clusters of integrin αIIbβ3 in pro-aggregatory platelets. Inhibition of integrin αIIbβ3 by 10E5 decreased binding of simoctocog alfa to platelets and subsequent PS exposure in a dose-dependent manner (Figure 2). Analysis of platelet binding and phenotype shift in patients with hemophilia A are ongoing. Conclusion: Simoctocog alfa demonstrated higher binding to activated platelets in vitro compared with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol, resulting in an increased phenotype shift of platelets from the pro-aggregatory to the pro-coagulant state. The increased binding of simoctocog alfa was associated with a phenotypic shift in platelets as evidenced by increased exposure of PS on the platelet membranes. The binding of simoctocog alfa to platelets was disrupted when integrin αIIbβ3 activation was inhibited, suggesting a role of integrin αIIbβ3 signaling following binding of FVIII to platelets. Variations in platelet binding and signaling between different rFVIIIs might impact their efficacy for the prevention of bleeds. Further experiments are ongoing to assess the impact of FVIII on the release of von Willebrand factor and thrombin generation by activated platelets, and to repeat the FVIII-binding experiments using platelets from healthy donors vs hemophilia A patients.

NXT007-mediated hemostatic potential is suppressed by activated protein C-catalyzed inactivation of activated factor V

BackgroundActivated protein C (APC) inactivates activated factor (F) V (FVa) and FVIIIa. NXT007, an emicizumab-based engineered therapeutic bispecific antibody, enhances the coagulation potential of FVIII-deficient plasma (FVIIIdef-plasma) to near normal levels. However, little is known about the effect of APC-induced inactivation in NXT007-mediated hemostatic function. ObjectivesTo investigate the contribution of APC-mediated reactions to NXT007-driven hemostasis. MethodsIn pooled normal plasma (PNP) or FVIIIdef-plasma spiked with NXT007 (10 μg/mL), effects of APC (0-16 nM) were measured using a thrombin generation assay (TGA). The direct effects of APC on cofactor activity of NXT007 or FVIIIa in a FXa generation assay were also measured. The FVdef-plasma and FV Leiden plasma (FVLeiden plasma) were preincubated with 2 anti-FVIII monoclonal antibodies (termed FVIII-depleted), and the APC effect in the presence of NXT007 in FVIII-depleted FVdef-plasma with the addition of exogenous FV (7.5-30 nM) or FVIII-depleted FVLeiden plasma was investigated. ResultsThe APC dose-dependent suppression effect in TGA of FVIIIdef-plasma spiked with NXT007 was similar to that of PNP. FXa generation with NXT007 was not impaired by the addition of APC, suggesting that the APC-induced reaction in TGA with NXT007 was attributed to the direct inactivation of FVa. The addition of APC to FVIII-depleted FVdef-plasma, along with NXT007 and various FV concentrations, showed a similar attenuation to PNP. The NXT007-driven thrombin generation in FVIII-depleted FVLeiden plasma was suppressed by APC, similar to the reaction in native FVLeiden plasma. ConclusionNXT007 did not impair APC-mediated downregulation of FVa in FVIIIdef-plasmas, regardless of the presence of FV mutation with APC resistance.

The Tfh/Tfr Pathway Is Pivotal in FVIII Inhibitor Development in Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIII null mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of Foxp3 + T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development. However, the role of Tfr cells in controlling anti-FVIII inhibitor development in vivo is still unclear. Here, we explored the activation and induction of Tfr cells and their functions in anti-FVIII inhibitor development in FVIII-immunized mice. We first characterized the features of activated Tfr cells in the spleen of immunized FVIII null mice by flow cytometry analysis. Activated Tfr cells were defined as CD4 +Foxp3 +CD44 +CD62L -CXCR5 +PD-1 +. Tfr cells express a high level of transcription factor BCL6 and costimulatory receptor ICOS. Activated Tfr cells also express a high level of costimulatory receptor CTLA4, which is similar to non-Tfr effector Treg cells, but CD25 expression is low on Tfr cells. FVIII immunization increases the frequency and the absolute number of activated Tfr cells in the spleen of mice that produce inhibitors. The percentage of activated CXCR5 hiPD-1 hi Tfr cells within the CD4 population significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88 ± 0.2 %, 0.33 ± 0.16 %, and 0.36 ± 0.2%, respectively). The Tfh/Tfr ratio also significantly increased in FVIII inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Interestingly, similar to Tfh cells as we reported before, the emergence of Tfr cells correlated with titers of anti-FVIII inhibitors in FVIII immunized mice. This indicates that a functional relationship exists between Tfr cell activation and FVIII inhibitor production after FVIII immunization. Using Tfr deficient Foxp3-Cre Bcl6 fl/fl (BCL6FC) mice, we determined the role of Tfr cells in inhibitor generation in vivo. Loss of Tfr cells led to significantly decreased FVIII inhibitor levels, with FVIII inhibitor titers in BCL6FC mice markedly lower than in wild-type (WT) mice upon recombinant human full-length FVIII (rhFVIII, Kogenate 200 U/kg/week IV x4) immunization (24 ± 16 and 131 ± 114 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and WT groups, suggesting that Tfr cells help generate neutralizing anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses. To further investigate the regulatory function of Tfr cells on FVIII immune responses, mice with phosphatase Pten deficiency in Treg cells (Foxp3-Cre Pten fl/fl [PtenFC]), a model with augmented Treg and Tfr cells, were immunized with rhFVIII and anti-FVIII antibody titers were measured at 1 week after the 4 th weekly immunization. Of note, FVIII inhibitor titers (8.06 ± 8.6 BU/ml) were significantly lower than those in WT controls under the same immunization protocol. There were significant positive correlations between the inhibitor titers and total anti-FVIII IgG in the WT and PtenFC groups but not in the BCL6FC group after rhFVIII immunization. To confirm the important role of the Tfh/Tfr pathway in FVIII inhibitor development, we used CD4-Cre Bcl6 fl/fl (CD4-Bcl6-cKO) mice, a model with Bcl6 deficiency in all CD4 + T cells. As expected, none of the CD4-Bcl6-cKO mice developed detectable levels of FVIII inhibitors, demonstrating that the transcription repressor BCL6 is pivotal for FVIII inhibitor development. In conclusion, we found that FVIII immunization induces Tfr cell activation and expansion. Tfr cells have a dual function in regulating FVIII immune responses in mice, and the Tfh/Tfr pathway is essential for FVIII inhibitor development in hemophilia A mice.

A Single Center Experience of 13 Episodes of Acquired Hemophilia A (2019 - 2023)

Background: Acquired Hemophilia A (AHA) is a rare bleeding disorder caused by neutralizing autoantibodies to factor VIII (FVIII). Treatment of AHA involves immunosuppression to eliminate the autoantibody and hemostatic management involving bypassing agents (BPAs). While the use of BPAs, including activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rFVIIa), is primarily derived from studies of congenital hemophilia with inhibitors, registry studies of AHA have also demonstrated the efficacy of rVIIa ( Baudo et al., Blood, 2012) and aPCC ( Borg et al., Haemophilia, 2015) ( Zanon et al., Br J Haem 2019) in this population. Given the changing treatment landscape, low incidence of AHA, and high morbidity and mortality associated with the disease, our study aims to describe a single institution's experience with treating AHA. Methods: We retrospectively reviewed the records of adult patients diagnosed with AHA at a single academic medical center between January 1, 2019, and June 30, 2023. Results: Eleven patients were treated for 13 distinct episodes of AHA, with one patient admitted for three discrete episodes of AHA with responses to therapy followed by relapses once lost to follow-up. The patients were predominantly white (63.7%, n=7) and female (63.7%, n=7), with a median age of 74 years and weight of 87 kg on admission (Table 1). One patient (9.1%) had concomitant autoimmune disease. No patients presented peripartum or with active malignancy, liver disease, or a family history of bleeding disorders. The median FVIII level on admission was 1% (IQR=1%, 3.5%), with a median anti-FVIII antibody titer of 27 BU (IQR=4.5 BU, 47 BU). Given mild bleeding presentation, Patient 2 was treated as an outpatient with immunosuppression only. All patients received aPCC as the first-line BPA. Patient 5 developed uncontrolled bleeding from an arterial line despite receiving aPCC and was transitioned to rFVIIa for two days before resuming aPCC once hemostasis was achieved. The median duration of aPCC therapy was eight days. The use of aPCC (FEIBA) instead of rFVIIa (NovoSeven) as the first-line BPA led to total estimated cost avoidance of $13.5 million (calculated using average wholesale price). No patients had thrombotic events after the diagnosis of AHA. Six episodes (46.1%) were treated with prednisone and rituximab. Six episodes (46.1%) were treated with prednisone and oral cyclophosphamide. One episode (7.7%) was treated initially with prednisone and cyclophosphamide but was eventually transitioned to rituximab following a lack of response and bleeding complications after two weeks. This patient was also subsequently treated with emicizumab for five months until FVIII activity was recovered. P neumocystis jirovic i i pneumonia prophylaxis was prescribed in eight episodes (61.5%), and antiviral prophylaxis was prescribed in one episode (7.7%). No infections were noted during the follow-up period, other than two documented cases of COVID-19, one of which required admission. The median time to achieve FVIII &amp;gt;50% was 5.1 weeks, with a median of 8.6 months of follow-up (Figure 1). Patient 11 relapsed twice, as described in Table 1. No other patient relapsed during the follow-up period. No patient deaths occurred during the follow-up period. Conclusions: We describe 13 episodes of AHA successfully treated at a single academic medical center between 2019 and 2023. aPCC was the first-line BPA administered in all patients, with significant cost savings relative to rFVIIa. Immunosuppression, including prednisone and either cyclophosphamide or rituximab, was used to successfully eliminate autoantibodies in all patients. One patient received emicizumab for ongoing hemostatic control until achieving improvement in FVIII activity. No significant infections, thrombotic events, or deaths were noted in this cohort.

Acquired Hemophilia a: A Single-Center Study of 165 Patients

Introduction and aim: Acquired hemophilia A (AHA) is a rare hemorrhagic disorder caused by the existence of factor VIII (FVIII) inhibitors with an estimated annual incidence of 1.5 per million people. AHA patients are characterized by exceeding bleeding of varying severity, while approximately 10% of AHA patients have no bleeding symptoms. It's a challenge to diagnose and manage AHA due to its rarity and heterogeneity. We conducted a retrospective study to report the characteristics and outcomes of AHA and enhance its recognition and management. Methods: Patients diagnosed and treated with AHA at the Blood Disease Hospital, Chinese Academy of Medical Sciences between July 1997 to December 2021 were retrospectively enrolled. Demographics, clinical characteristics, treatments, and outcomes were collected. All Patients were followed immediately from admission to our center and ended in March 2022. Bleeding severity was assessed retrospectively in March 2022 by bleeding score (BS), a summary score aggregated by the International Society on Thrombosis and Hemostasis/ Scientific and Standardization Committee (ISTH/SSC) bleeding assessment tool (BAT) (2010 edition). Results: A total of 165 AHA patients admitted to our center were enrolled. The median age at diagnosis was 45 (10-90) years. Overall, a female preponderance (106/165, 64.2%) was observed in this cohort. Notable, the proportion of male patients increased with age. Most patients (126/165, 76.4%) were idiopathic with no underlying diseases discovered. Of the 39 (23.6%) patients with associated diseases, 12 patients had autoimmune diseases, 16 patients had infections, eight patients had malignancies (all solid tumors) and three patients were secondary to pregnancy. All patients presented at least one bleeding episode. Subcutaneous bleeding was most common affecting 84.8% (140/165) of patients, while hemarthrosis was infrequent (n=11, 6.7%). The median time from bleeding to be diagnosed as AHA was delayed (30, 2-5650 days). Anti-FVIII antibody titer and FVIII level showed no correlation. A significant difference in FVIII activity was observed between patients with anti-FVIII antibody titer &amp;lt; 20 BU/ml and ≥ 20 BU/ml. The BS varied among all patients ranging from 2 to 12 with a median of 4. A significant difference in BS was observed among the mild (FVIII: C &amp;gt; 5 IU/dl), moderate (1 IU/dl &amp;lt; FVIII: C ≤ 5 IU/dl), and severe (FVIII: C ≤ 1 IU/dl) AHA patients (p &amp;lt; 0.05). The same conclusion was also drawn between patients with low anti-FVIII antibody titer (&amp;lt; 20 BU/ml) and high antibody titer (≥ 20 BU/ml). BS was not correlated with APTT (r=0.-0.04, 95% CI (-0.19-0.12), p &amp;gt; 0.05). Hemostatic treatment was applied to 129 (78.2%) AHA patients. Of the 163 patients who received immunosuppressive therapy (IST), 22 (13.5%) received steroids alone, with an 82.4% complete remission (CR) rate, 50 (30.7%) received steroids plus cyclophosphamide (CTX), with an 85.0% CR rate, 80 (49.1%) received rituximab-based therapy, which includes rituximab plus steroids, rituximab plus steroids and CTX, and rituximab plus steroids and other immunosuppressants, with a 93.3% CR rate (Figure 1A). There were seven deaths. Causes of death were bleeding (n=3), underlying disease (n=1), pulmonary infection (m=1), and unknown (n=2). FVIII inhibitor titer ≥ 20 BU/ml was the only significant poor prognostic factor for CR (HR 0.56; 95% CI, 0.37-0.85; p &amp;lt; 0.01). In addition, patients with FVIII inhibitor titers ≥ 20 BU/ml needed more time to achieve CR (130 days vs. 35 days; p &amp;lt; 0.01) and showed a lower CR rate (77.6% vs. 94.9%, p &amp;lt; 0.01) than those with FVIII inhibitor titers &amp;lt; 20 BU/ml (Figure 1B). Conclusions: Delayed diagnosis of AHA patients is a severe issue. The rituximab-based regimen is more likely to achieve a CR in AHA patients. The only poor predictor of CR for AHA patients was FVIII inhibitor titer ≥ 20 BU/ml.

Two-center validation of assays for the detection of binding and neutralizing anti-factor VIII antibodies.

Patients with hemophilia A treated with coagulation Factor VIII (FVIII) products are at risk for developing anti-FVIII antibodies. The ABIRISK Consortium aimed to provide knowledge on the formation and detection of anti-drug antibodies against biopharmaceutical products, including FVIII. Accordingly, standardized and validated assays for the detection of binding (total) and neutralizing antibodies are needed. Two-center validation of an ELISA for the detection of total FVIII-binding IgG-antibodies and Nijmegen-Bethesda assays for the quantification of FVIII-neutralizing antibodies according to consensus validation guidelines. Validation of assays at both sites was done according to published recommendations and included preanalytics, the determination of key assay parameters, including cut-points, assay sensitivity, precision, and FVIII interference. The validated assays reproducibly detected FVIII-binding and -neutralizing antibodies with comparable performance in both laboratories. Floating screening cut-points were established for both assays. Determined mass-based sensitivity of both assays (all values ≤66ng/mL) complied with the minimum sensitivity for the detection of anti-drug antibodies as recommended by the FDA (<100ng/mL). Intra- and inter-assay coefficients of variation did not exceed 25%. Assay validation further revealed that pre-analytical heat treatment led to potentially false-positive ELISA results, while up to 0.15 IU/mL, residual FVIII showed no significant impact. Overall, good agreement of results was found for patient samples analyzed at both study sites. Comprehensive validation of different anti-FVIII-antibody assays in two laboratories gave novel insights into the impact of pre-analytical sample treatment as well as the comparability of test results generated by the use of methodically different assays.

Von Willebrand factor modulates immune complexes and the recall response against factor VIII in a murine hemophilia A model

AbstractAchieving tolerance toward factor VIII (FVIII) remains an important goal of hemophilia treatment. Up to 40% of patients with severe hemophilia A (HA) develop neutralizing antibodies against FVIII, and the only proven treatment to achieve tolerance is infusion of FVIII over prolonged periods in the context of immune tolerance induction. Here, we addressed the role of von Willebrand factor (VWF) as a modulator of anti-FVIII antibody effector functions and the FVIII-specific recall response in an HA mouse model. Analytical ultracentrifugation was used to demonstrate formation of FVIII-containing immune complexes (FVIII-ICs). VWF did not fully prevent FVIII-IC formation but was rather incorporated into larger macromolecular complexes. VWF prevented binding of FVIII-ICs to complement C1q, most efficiently when it was preincubated with FVIII before the addition of antibodies. It also prevented binding to immobilized Fc-γ receptor and to bone marrow–derived dendritic cells. An in vitro model of the anti-FVIII recall response demonstrated that addition of VWF to FVIII abolished the proliferation of FVIII-specific antibody-secreting cells. After adoptive transfer of sensitized splenocytes into immunocompetent HA mice, the FVIII recall response was diminished by VWF. In summary, these data indicate that VWF modulates the formation and effector functions of FVIII-ICs and attenuates the secondary immune response to FVIII in HA mice.

Mice possess a more limited natural antihuman factor VIII antibody repertoire than humans that is produced disproportionately by marginal zone B cells

BackgroundOne-third of patients with severe hemophilia A develop neutralizing antibodies to the factor VIII (FVIII) protein in response to intravenous replacement therapy. Patients may also generate natural, nonneutralizing antibodies to FVIII before FVIII exposure. These patients are at increased risk of developing neutralizing antibodies to FVIII. However, natural anti-FVIII antibodies are also present in healthy human donors. ObjectivesTo further characterize the natural antihuman (h) FVIII antibody repertoire in mice and humans. MethodsAn in-house ELISA was developed using a purified polyclonal immunoglobulin (Ig) standard to quantify anti-hFVIII Ig in cell culture supernatant or plasma from mice (wild-type and FVIII-/-) and adult human donors. ResultsAll naïve wild-type and FVIII-/- mice, as well as healthy human donors, possess natural anti-hFVIII antibodies. Mice only have natural anti-hFVIII IgM, which is present in germ-free mice, suggesting that they are germline encoded. Although murine marginal zone B cells (MZBs) contribute 44% to all circulating natural IgM, they contribute disproportionately to the anti-hFVIII IgM repertoire (82%). This naturally occurring murine MZB-derived IgM is not B-domain specific and is reduced by intravenously administered hFVIII, suggesting that it may form immune complexes immediately upon hFVIII administration. Natural anti-hFVIII antibodies of IgG, IgM, and IgA isotypes can be detected in adult human donors. There were increased levels of B-domain-favoring anti-hFVIII IgG in 14% of healthy donors, which were markedly different from the rest of the “low-titer” population. ConclusionsThere is a preponderance of natural anti-hFVIII antibodies in both mice and healthy adult human donors.

Anti-FVIII antibodies in Black and White hemophilia A subjects: do F8 haplotypes play a role?

The most common complication in hemophilia A (HA) treatment, affecting 25% to 30% of patients with severe HA, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 haplotypes H1 to H5 are defined by nonsynonymous single-nucleotide polymorphisms encoding sequence variations at FVIII residues 1241, 2238, and 484. Haplotypes H2 to H5 are more prevalent in individuals with Black African ancestry, whereas 80% to 90% of the White population has the H1 haplotype. This study used an established multiplex fluorescence immunoassay to determine anti-FVIII antibody titers in plasma from 394 individuals with HA (188 Black, 206 White), measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3/H5, and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic assay and linear B-cell epitopes characterized using peptide microarrays. FVIII-reactive antibodies were readily detected in most individuals with HA, with higher titers in those with a current inhibitor, as expected. Neither total nor inhibitory antibody titers correlated with F8 haplotype mismatches, andpeptides with D1241E and M2238V polymorphisms did not comprise linear B-cell epitopes. Interestingly, compared with the full-length FVIII products, the BDD-FVIII proteins were markedly more reactive with plasma antibodies. The stronger immunoreactivity of BDD-FVIII suggests that B-domain removal might expose novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.