Development Of Inhibitory Antibodies Research Articles

The prophylaxis of severe hemophilia A with inhibitors in children in the Republic of Belarus: a 12-year experience

The development of inhibitory antibodies against FVIII is the most serious complication associated with the use of FVIII concentrates in hemophilia A patients. There is a need for more research on measures that could reduce the risk of inhibitor formation in previously untreated patients (PUPs) with severe hemophilia A. The purpose of this study was to determine the effectiveness of the prevention of clotting inhibitor development in PUPs (or minimally treated patients) with severe hemophilia A by administering plasma-derived factor VIII concentrate (pdFVIII) at a dose of 25 IU/kg once a week for a year. The study was approved by the Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). Between 2010 and 2022, 56 boys were newly diagnosed with severe hemophilia A. Twenty-one of them received pdFVIII as on-demand treatment to stop bleeding (Group 1). Thirty-five boys received pdFVIII at a dose of 25 IU/kg body weight once a week during the first 50 weeks of treatment for the prevention of inhibitor development (Group 2). The administration of pdFVIII at a dose of 25 IU/kg once a week in the PUPs (or minimally treated patients) contributed to a decrease in the cumulative incidence of inhibitors to 15.9 ± 7.7% (4 out of the 35 patients who had been treated prophylactically) compared with 43.7 ± 11.8% (8 out of the 21 patients who had received hemostatic therapy to stop bleeding) (log-rank test, p = 0.041). Thus, the administration of pdFVIII concentrate at a dose of 25 IU/kg once a week for the first 50 weeks of treatment lead to a decrease (p = 0.009) in the cumulative incidence of inhibitors against the administered coagulation factor VIII to 15.9 ± 7.7%.

Factor IX antibodies and tolerance in hemophilia B in the Nordic countries – The impact of F9 variants and complications

IntroductionThe development of inhibitory antibodies (inhibitors) in persons with hemophilia B (PwHB) causes significant morbidity. Data on the impact of the F9 variant and immune tolerance induction (ITI) outcome are limited.The aim of this study was to investigate the presence of neutralizing and non-neutralizing antibodies (NNA) in severe hemophilia B (HB) and to evaluate ITI outcome and complications in relation to the pathogenic F9 variant. Materials and methodsPersons with severe HB in the Nordic countries were enrolled and information on F9 variants, inhibitors, ITI and complications were collected. Analyses of anti-FIX antibodies with a fluorescence-immunoassay (xFLI) and an ELISA method were conducted. ResultsSeventy-nine PwHB were enrolled. Null variants were seen in 33 (42 %) PwHB and 12 (15 %) had a current or former inhibitor. Eleven (92 %) of the inhibitor patients had experienced allergic manifestations and three (25 %) nephrotic syndrome. Of 10 PwHB with at least one ITI attempt, eight (80 %) were considered tolerant at enrolment. Immunosuppression was included in seven of eight successful or partially successful attempts. Five PwHB had at least one ITI failure before a successful or partially successful ITI. No NNA could be identified. ConclusionA high proportion of severe F9 gene defects among persons with severe HB in the Nordic countries may explain the observed relatively high prevalence of inhibitors. ITI success was independent of the F9 variant and attained despite allergic manifestations and previous ITI failures. Inclusion of immunosuppression tentatively enhances the chances of ITI success. No NNA were observed.

Designing ATHN 7: Safety, effectiveness and practice of treatment of people with haemophilia in the United States through a natural history cohort study

Abstract Background Haemophilia A and B are X-linked inherited bleeding disorders, resulting in the deficiency of clotting factor VIII and IX, respectively. Since the introduction of recombinant clotting factor concentrates in the early 1990s, the major safety concern for haemophilia therapy has been the development of inhibitory antibodies, or inhibitors. Over the recent past, new therapies for the treatment and prevention of bleeding have received regulatory approval or are under study. Objective ‘ATHN 7: A Natural History Cohort Study of the Safety, Effectiveess, and Practice of Treatment for People with Hemophilia is designed to determine the safety of current haemophilia therapies when used for participants with haemophilia with or without inhibitors. Secondarily, ATHN 7 will describe the real-world effectiveness of current therapies by assessing bleeding rate and location, therapy utilisation, adherence, and patient-reported outcomes. Methods This longitudinal, observational cohort study by the American Thrombosis and Hemostasis Network (ATHN) will follow participants with haemophilia with or without inhibitors for four years from the time of enrolment. Each participant is assessed every three months. All data are collected into ATHN Systems. The primary outcome measure is the incidence of safety events as defined by the European Haemophilia Safety Surveillance (EUHASS) programme. Effectiveness will be described based on annualised bleeding rate, therapy utilisation, adherence, and patient-reported outcomes. Conclusion As the first product-agnostic, real-world study of haemophilia therapy in the United States, ATHN 7 collects data to determine current intervention safety and effectiveness. Based on this success, ATHN will continue to collect these data longitudinally through the ATHN Transcends study.

Immune tolerance induction in the era of emicizumab - still the first choice for patients with haemophilia A and inhibitors?

The development of inhibitory antibodies is a severe complication of clotting factor replacement therapy in patients with severe haemophilia A (HA). Current World Federation of Hemophilia (WFH) guidelines for haemophilia care indicate that eradication of inhibitors is best achieved through immune tolerance induction (ITI) therapy. The European Collaborative Haemophilia Network conducted a survey to determine whether ITI is still used in the routine management of patients with HA, and whether the availability of emicizumab prophylaxis has influenced treatment decisions. The survey was conducted in late 2020/early 2021 in 18 centres representing 17 countries in the Europe/Middle East region treating a total of 4955 patients, and included sections specific to patient and centre demographics, treatment protocols (both ITI and prophylactic), inhibitor development and initiation of ITI, treatment success, and the incidence of adverse events. While our results indicate that ITI can still be considered a mainstay of treatment for patients with HA with inhibitors, less than daily dosing of ITI in combination with emicizumab prophylaxis is becoming commonplace across the spectrum of disease severity, with initiation being guided by bleeding patterns. The most frequently cited reasons for not initiating emicizumab prophylaxis were availability or reimbursement issues. ITI remains a mainstay for haemophilia treatment of patients with HA with inhibitors, but emicizumab has become a preferred first-line approach to protect against bleeds and represents an alternative to burdensome ITI in certain patient groups.

Serum TNF-α Level as a Possible Predictor of Inhibitor Levels in Severe Hemophilia A.

Background The development of factor VIII (FVIII) inhibitor in patients with hemophilia A (PWHA) is a great challenge for hemophilia care. Both genetic and environmental factors led to complications in PWHA. The development of inhibitory antibodies is usually induced by the immune response. Tumor necrosis factor α (TNF-α), one of the cytokines, might contribute to its polymorphism. In this study, we investigated the clinical factors, level of serum TNF-α, and polymorphism of c.−308G > A TNF − α gene in inhibitor development in severe PWHA. Methods A cross-sectional study was conducted among all PWHA in West Java province. The clinical parameters, FVIII, FVIII inhibitor, and serum TNF-α level were assessed. The genotyping of −380G > A TNF-α gene polymorphism was performed using polymerase chain reaction and Sanger sequencing. Results Among the 258 PWHA, 216 (83.7%) were identified as severe PWHA. The FVIII inhibitor was identified in 90/216 (41.6%) of severe PWHA, consisting of 45 high-titer inhibitors (HTI) and 45 low-titer inhibitors (LTI). There was a significant correlation between serum TNF-α level and the development of HTI (p = 0.043). The cutoff point of serum TNF-α level, which can be used to differentiate between HTI and LTI, was 11.45 pg/mL. The frequency of FVIII replacement therapy was significant only in HTI of severe PWHA regarding serum TNF-α level (p = 0.028). There is no correlation between polymorphisms of −380G > A TNF-α gene and inhibitor development (p = 0.645). Conclusions The prevalence of FVIII inhibitor in severe PWHA in West Java, Indonesia, was 41.6%. The frequency of replacement therapy is a risk factor for inhibitor development. Serum TNF-α level might be used to differentiate between high and low inhibitor levels in severe hemophilia A, and this might support decision making regarding treatment options for inhibitor in severe hemophilia A.

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.

Generation of FVIII-Specific Tregs Expressing a Chimeric Antigen Receptor (CAR) to Suppress Inhibitor Development in Hemophilia a Mice

Introduction: The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is a critical complication in the treatment of hemophilia A, as hemostasis can no longer be re-established by FVIII replacement therapy. Cellular Treg therapy is an attractive candidate for targeted treatment of B and T cells involved in inhibitor formation, as it can suppress both cell types through direct and indirect mechanisms. Here, we generated antigen specific Tregs by engineering and retrovirally transducing a chimeric antigen receptor (CAR) molecule with specificity to human FVIII. Antigen recognition via the single chain antibody variable regions triggers signaling via the CD28 and CD3z co-stimulatory domains, which induces CAR-Treg activation and proliferation independent of MHC recognition. We show that human FVIII specific CAR-Tregs with second generation mouse signaling domains are activated and proliferate in response to antigen recognition, which can cause contact dependent or cytokine dependent suppression of inhibitor producing cells. Methods:A single chain variable fragment specific for human FVIII was cloned (Creative Biolabs, NY) out of an EBV transformed B cell from a hemophilia A patient (BO2C11), which produces IgG4 directed against residues 2125-2332 of huFVIII, corresponding to the carboxy-terminal end of C1 and the complete highly immunogenic C2 domain. This was fused to a 2nd generation CAR construct expressing CD28 and CD3ζ signaling molecules and packaged in a retroviral system (pMys-IRES-eGFP). CD3/CD28 activated polyclonal Tregs were retrovirally transduced to generate FVIII CAR-Tregs. Binding to Fc-FVIII was tested using an A647 conjugated Fc antibody. Upregulation of activation and exhaustion markers, specifically CD69, CD44, LAP, GARP and PD1 was tested 24 h after stimulation with either Fc-FVIII or B domain deleted (BDD) FVIII. Proliferation of CellTrace Violet labeled and FVIII stimulated CAR-Tregs was tested both in vitro and in vivo upon stimulation with BDD FVIII. 1x106 GFP+ FVIII CAR-Tregsorted cells were adoptively transferred into F8 e16−/− hemophilia A mice, and recipients were challenged, starting 1 day later, with weekly IV injections of 1 IU BDD-FVIII for 4 weeks. Plasma was tested for inhibitor formation at 4 and 8 weeks using the Bethesda assay and IgG1 ELISA. Results:FVIII CAR-Tregs bound Fc-FVIII (Eloctate) in a specific manner, with no binding observed to control Fc-FIX (Alprolix). Stimulation for 24 hours with either Fc-FVIII or BDD-FVIII resulted in upregulation of activation and exhaustion markers such as CD69, CD44, GARP and PD-1. No non-specific activation with the unrelated Fc-FIX antigen was observed. CellTrace Violet labeled CAR-Treg and CAR transduced conventional T cells underwent antigen-specific proliferation upon 72 hours of in vitro stimulation with FVIII. In vivo, persistence of adoptively transferred CAR-Tregs was observed only when antigen was concurrently administered. We are currently testing for the suppression of inhibitor development in BALB/c F8 e16−/− hemophilia A mice following adoptive immunotherapy with FVIII CAR-Tregs and continued weekly administrations of BDD-FVIII. Conclusions: Here, we demonstrate that CAR-Tregs directed to human FVIII show antigen-specific binding, activation and proliferation. We found persistence of adoptively transferred FVIII CAR-Tregs in response to antigen stimulation in mice. The suppressive activity of FVIII CAR-Treg on inhibitory antibody formation is now being evaluated in hemophilia A mice that are given factor replacement therapy with FVIII. CAR-Tregs represent an effective way to generate a large pool of antigen-specific cells, with no requirement for MHC restriction. DisclosuresBiswas:Bayer Hemophilia Awards Program: Research Funding. Herzog:Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties.

HLA-DRB1-Peptide Binding Prediction and Risk of Inhibitor Development in Non-Severe Hemophilia a

Background: Anti-factor VIII (FVIII) inhibitory antibody development (inhibitors) occurs in up to 13% of patients with non-severe hemophilia A (HA) and is known to be associated with receipt of intensive FVIII treatment (Eckhardt et al. 2013) and F8 mutations such as p.Arg612Cys. Inhibitor development is a T-cell dependent process requiring presentation of a novel peptide by HLA-class II on the surface of antigen presenting cells. Computer algorithms can be used to model the binding affinity of a peptide for a specific HLA type (Zhang et al. 2012). The strength of association between computer-modeled HLA-DRB1 and mutation-specific FVIII peptide binding and inhibitor development in non-severe HA with known F8 and HLA-DRB1 genotype is unknown. Methods: In a cohort of non-severe HA (FVIII 1-40%) collected as part of a case-control study (Kempton et al. 2010), subjects with a missense mutation who had consented to participate in a repository for future research were evaluated. Case subjects had an inhibitor titer > 1.0 BU/ml on two occasions or on one occasion with subsequent immune tolerance induction. Control subjects had prior FVIII exposure and no history of inhibitor ( Results: Fifty-seven subjects (case n=20 and control n=37, 56% of the original study sample) were included in this analysis. The median age (case 37 years vs control 27 years) and baseline FVIII (case 8.0 IU/dl vs control 5.5 IU/dl) were similar between groups. Cases and controls were also similar with respect to race, ethnicity, prior FVIII exposure, family history of inhibitor, FVIII product class used (recombinant vs. plasma-derived), and being HIV or HCV antibody positive. F8 mutations found in more than 1 case included p.Arg612Cys (case=6; control=1), p.Asn1941Ser (case=5; control=1), and p.Arg2169His (case=3; control=1). HLA alleles found both in more than 10% of cases and more frequently in cases than controls included HLA-DRB1*15:01 (case =7 [18%], control =7 [9%], OR 2.3 [95% CI 0.5-10.8]), HLA-DRB1*13:02 (case=5 [13%], control=4 [5%], OR 2.8 [95% CI 0.5-16.4]); HLA-DRB1*11:01 (case=4 [10%], control = 3 [4%], OR 3.0 [95% CI 0.4-20.2]). The median number of novel HLA-DRB1 allele/peptide combinations among cases and controls is shown in Table 1. The association of having > 10 novel HLA-DRB1 allele/peptide combinations with inhibitor development is shown in Table 2. Conclusions: Having more than 10 FVIII peptides (>1/3 of the potential FVIII peptides) that are able to bind HLA-DRB1 and present a novel surface is associated with inhibitor development independent of age, family history of inhibitor, and intensive FVIII treatment. The presentation of novel HLA-DRBI peptide combinations appears to be an intermediary between specific high-risk F8 mutations and inhibitor development. Disclosures Kempton: Genentech: Membership on an entity9s Board of Directors or advisory committees.

Reprogrammed CD4+ T Cells That Express FoxP3+ Effectively Control Inhibitory Antibody Development in Hemophilic Mice

Introduction: CD4+CD25+FoxP3+ regulatory T cells (Treg) can suppress a range of immune functions, including the activation and proliferation of conventional CD4+ T cells. Inhibitory antibody development to clotting factor replacement therapy in the X-linked disorder hemophilia is dependent on T cell help. Therefore, Tregs play a crucial role in tolerance to the therapeutic replacement protein. Adoptive immunotherapy with autologous or donor Tregs have shown promise in several clinical trials for autoimmune disorders and in transplant conditions. However, antigen-specific Tregs are extremely rare and difficult to isolate. Ectopic overexpression of FoxP3 in conventional CD4+ T cells from hemophilia patients can potentially create a large pool of Tregs, enriched for antigen specificity. Here we show that adoptive immunotherapy with FoxP3 overexpressing CD4+ T cells can control immune responses to clotting factor replacement therapy in a mouse model of hemophilia A. Methods:BALB/c F8 e16−/− hemophilia A mice were repeatedly injected with recombinant B-domain deleted (BDD) FVIII to generate high inhibitory antibody (inhibitor) titers and FVIII-specific T cells. CD4+CD25- conventional T cells were isolated and transduced with pMIGR-eGFP retroviral vector encoding the murine FoxP3 gene (pMIGR-FoxP3-eGFP). 2x106 GFP+ FoxP3+ sorted cells were adoptively transferred into F8 e16−/− mice, and recipients were challenged 1 day later with weekly IV injections of 1 IU BDD-FVIII for 4 weeks. Plasma was tested for inhibitor/antibody formation at 4 and 8 weeks using the Bethesda assay and ELISA. Furthermore, mice with pre-existing inhibitors received adoptive transfer of a single or multiple doses of 2x106 FoxP3+ sorted cells to assess inhibitor reversal. FoxP3+GFP+ sorted cells were adoptively transferred into naïve mice and re-isolated 2 days later to test for phenotypic marker expression, namely CD25, FoxP3, GITR, CD39, CTLA-4, Neuropilin-1, TNFR2, CD127, and CD62L. Antigen-specific suppressive activity was assayed in vitro using FoxP3 transduced CD4+ T cells from transgenic DO11.10 Rag-/- mice with TCR specificity for ovalbumin (OVA). The proliferation of CellTrace Violet labeled DO11.10 Rag-/- responder splenocyteswas calculated in response to stimulation with OVA323-339 peptide. Results:Retroviral transduction of CD4+CD25- cells with the murine FoxP3 gene resulted in a population of cells that phenotypically resemble peripheral Tregs. FoxP3 transduced Tregs underwent antigen specific proliferation in vivo and stably maintained FoxP3 expression in proliferating cells. FoxP3 transduced OVA-specific CD4+ T from DO11.10 Rag-/- mice suppressed the proliferation of transgenic responder cells in response to OVA323-339 peptide antigen. CD4+ T cells transduced with empty vector or freshly isolated natural Tregs were unable to suppress proliferation. Adoptively transferred FoxP3 transduced cells from BDD-FVIII immunized donor mice completely prevented the induction of inhibitors following challenge with repeat injections of BDD-FVIII. FoxP3 transduction of naïve donors (i.e. not FVIII antigen-experienced) were inferior in suppressing inhibitor formation. We are currently testing for reversal / reduction of established inhibitory antibodies with a single or repeat doses of FoxP3 transduced BDD-FVIII immunized donor mice. Conclusions and future directions: Treg based therapies using ex vivo expanded recipient-derived Tregs are currently in clinical trials as primary or adjunct treatment for allograft rejection and autoimmune disease. Here, forced expression of FoxP3 into antigen-specific conventional CD4+ T cells by retroviral gene transfer resulted in a population of antigen-specific cells with phenotypic characteristics of Tregs. These cells effectively suppressed formation of inhibitors in BALB/c F8 e16−/− hemophilia A mice for a sustained 2-month period. We will further explore the mechanism of suppression, including the possibility of endogenous Treg induction via infectious tolerance. DisclosuresBiswas:Bayer Hemophilia Awards Program: Research Funding. Herzog:Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties.

Haemophilia.

Haemophilia A and B are rare congenital, recessive X-linked disorders caused by lack or deficiency of clotting factor VIII (FVIII) or IX (FIX), respectively. The severity of the disease depends on the reduction of levels of FVIII or FIX, which are determined by the type of the causative mutation in the genes encoding the factors (F8 and F9, respectively). The hallmark clinical characteristic, especially in untreated severe forms, is bleeding (spontaneous or after trauma) into major joints such as ankles, knees and elbows, which can result in the development of arthropathy. Intracranial bleeds and bleeds into internal organs may be life-threatening. The median life expectancy was ~30 years until the 1960s, but improved understanding of the disorder and development of efficacious therapy based on prophylactic replacement of the missing factor has caused a paradigm shift, and today individuals with haemophilia can look forward to a virtually normal life expectancy and quality of life. Nevertheless, the potential development of inhibitory antibodies to infused factor is still a major hurdle to overcome in a substantial proportion of patients. Finally, gene therapy for both types of haemophilia has progressed remarkably and could soon become a reality.

Prediction of hemophilia A severity using a small-input machine-learning framework

Hemophilia A is a relatively rare hereditary coagulation disorder caused by a defective F8 gene resulting in a dysfunctional Factor VIII protein (FVIII). This condition impairs the coagulation cascade, and if left untreated, it causes permanent joint damage and poses a risk of fatal intracranial hemorrhage in case of traumatic events. To develop prophylactic therapies with longer half-lives and that do not trigger the development of inhibitory antibodies, it is essential to have a deep understanding of the structure of the FVIII protein. In this study, we explored alternative ways of representing the FVIII protein structure and designed a machine-learning framework to improve the understanding of the relationship between the protein structure and the disease severity. We verified a close agreement between in silico, in vitro and clinical data. Finally, we predicted the severity of all possible mutations in the FVIII structure – including those not yet reported in the medical literature. We identified several hotspots in the FVIII structure where mutations are likely to induce detrimental effects to its activity. The combination of protein structure analysis and machine learning is a powerful approach to predict and understand the effects of mutations on the disease outcome.

Real-World Clinical Management of Patients with Congenital Hemophilia and Inhibitors: Interim Analysis of the FEIBA Global Outcome Study (FEIBA GO)

Introduction and Objective: Development of inhibitory antibodies is a major complication of factor replacement therapy in patients with congenital hemophilia A or B. The FEIBA Global Outcome study (FEIBA GO) assessed the long-term safety and real-world effectiveness of activated prothrombin complex concentrate (aPCC; Baxalta US Inc, a Takeda company, Lexington, MA, USA) as prophylaxis or on-demand treatment in patients with congenital hemophilia A or B with inhibitors (PwHI) across different clinical settings. Methods: FEIBA GO (EUPAS6691) is a post-authorization, prospective, observational, multicenter cohort study. Male PwHI diagnosed before study entry and prescribed treatment with aPCC as part of routine clinical practice were followed over 4 years; treatment regimens were prescribed at the physician's discretion. Ethics approval and patient consent were obtained. These data report on an interim analysis (data cutoff, May 16, 2019) on the annualized bleeding rate (ABR) calculated per patient-year of follow-up and aPCC consumption in patients receiving aPCC prophylaxis. Results: Enrollment was started on September 3, 2014 and completed on December 31, 2017. Fifty-one enrolled PwHI have received aPCC prophylaxis (n=37) or on-demand (n=14) treatment from 26 sites in 11 countries (hemophilia A: n=50, hemophilia B: n=1; median [range] age at baseline: 17 [2-71] years). As of May 2019, mean±SD (median, range) ABR was 7.2±8.2 (5.7, 0-30) per patient-year for the 14 patients with ≥2 to <4 years' study follow-up (mean±SD: 3.0±0.6 years). In the 3 patients with ≥4 years of study follow-up (mean±SD: 4.0±0.03 years), mean±SD (median, range) ABR was 14.7±25.3 (0.2, 0-44) per patient-year. Patients received a variety of prophylaxis regimens; data on dosages per infusion and number of weekly infusions administered are provided in the Table. Conclusions: This interim analysis describes the real-world use and effectiveness of aPCC prophylaxis and supports previous data on the prevention of bleeding events in PwHI. The data also highlight the variety of dosing regimens reflecting the real-world use of aPCC prophylaxis in clinical practice. Although patient numbers were small, these data suggest that increased weekly doses are used in patients with more severe bleeding phenotypes. Figure 1 Disclosures Hermans: Kedrion:Speakers Bureau;Octapharma:Consultancy, Speakers Bureau;Roche:Consultancy, Speakers Bureau;Pfizer:Consultancy, Research Funding, Speakers Bureau;Novo Nordisk:Consultancy, Speakers Bureau;LFB:Consultancy, Speakers Bureau;Shire, a Takeda company:Consultancy, Research Funding, Speakers Bureau;Sobi:Consultancy, Research Funding, Speakers Bureau;Bayer:Consultancy, Research Funding, Speakers Bureau;EAHAD:Other;WFH:Other;CSL Behring:Consultancy, Speakers Bureau;CAF-DCF:Consultancy, Speakers Bureau;Biogen:Consultancy, Speakers Bureau.Négrier:CSL Behring, Octapharma, Shire/Takeda, Sobi:Research Funding;CSL, F. Hoffmann-La Roche Ltd, Sobi:Other: Travel support;Bayer, Biomarin, CSL Behring, Freeline, LFB, Novo Nordisk, Octapharma, Pfizer, F. Hoffmann-La Roche Ltd, Sanofi, Shire/Takeda, Sobi, Spark:Consultancy.Holme:Sobi:Honoraria, Research Funding;Bayer:Honoraria, Research Funding;CSL Behring:Honoraria;Novo Nordisk:Honoraria;Octapharma:Research Funding;Pfizer:Honoraria, Research Funding;Shire, a Takeda company:Honoraria, Research Funding.Escuriola:Grifols:Honoraria;CSL Behring:Consultancy, Honoraria, Research Funding;Biotest:Honoraria, Research Funding;Biomarin:Honoraria;Bayer:Honoraria;Kedrion:Honoraria;Shire, a Takeda company:Honoraria;Octapharma:Consultancy, Honoraria, Research Funding;Novo Nordisk:Consultancy, Honoraria;Roche:Honoraria;Sobi:Honoraria, Research Funding.Cid:Roche:Consultancy, Honoraria;Sobi:Consultancy, Honoraria;Novo Nordisk:Honoraria;Shire, a Takeda company:Honoraria.Kemenyash:Takeda Pharmaceutical International AG:Current Employment, Current equity holder in publicly-traded company.Botha:Takeda Pharmaceutical International AG:Current Employment, Current equity holder in publicly-traded company.Cano-Garcia:Sanofi Genzyme:Current Employment, Current equity holder in publicly-traded company;Shire, a Takeda company:Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months.Windyga:Sanofi:Honoraria, Research Funding;Alexion:Honoraria, Research Funding;Alnylam:Honoraria, Research Funding;Baxalta/Shire, a Takeda company:Honoraria, Research Funding;Bayer:Honoraria, Research Funding;Siemens:Honoraria, Research Funding;Sobi:Honoraria, Research Funding;Werfen:Honoraria, Research Funding;CSL Behring:Honoraria, Research Funding;Ferring Pharmaceuticals:Honoraria, Research Funding;Novo Nordisk:Honoraria, Research Funding;Octapharma:Honoraria, Research Funding;Rigel Pharmaceuticals:Honoraria, Research Funding;Roche:Honoraria, Research Funding.

Disentangling the Effects of HLA DRB1*15:01 and DQB1*06:02 to Establish the True HLA Risk Allele for Inhibitor Development in the Treatment of Hemophilia A

The HLA haplotype DRB1*15:01/DQB1*06:02 has been shown to be associated with the development of inhibitory antibodies ("inhibitors") against therapeutic FVIII (tFVIII) proteins in Hemophilia A (HA) patients. Moreover, this same haplotype has also been implicated with an increased risk for the development of multiple sclerosis, narcolepsy, and drug-induced-hypersensitivity and -liver-injury. Recent reports in the literature have indicated that the alleles of this haplotype may have differential effects on immunogenicity despite the fact that they are in strong linkage disequilibrium (LD). Thus, the question arises as to which of the two constituent alleles of this haplotype is the true HLA risk allele. To address this question we analyzed peptidomic profiling data on HLA-class-II (HLAcII)-presented peptides from dendritic cell (DC)-protein processing and presentation assays (PPPAs) performed in two independent experiments performed by Peyron et al. (2018) and Diego et al. (2020) which reported results on both the HLA-DQ and -DR isomers. The tFVIII in both experiments is full-length recombinant FVIII (FL-rFVIII). We performed log-linear mixed model analyses under two models, where the dependent variable in both models is the logarithm of the expected peptide count. Under Model 1, we analyzed in the fixed effect component of the model a single HLA allele predictor variable consisting of 10 levels represented by distinct DRB1 and DQB1 alleles (4 and 6 levels, respectively). Under this model, we analyzed in the random effects component of the model variables for individuals (8 and 9 levels from Peyron et al. and Diego et al., respectively), experiments (2 levels for Peyron et al. and Diego et al.), and HLA isomers (2 levels for DQ and DR). The random effects component of the model serves to reduce the error (or "noise") in our estimation of the predictive effect of HLA alleles for the peptide counts by accounting for the clustering due to individuals, experiments, and isomers. This approach therefore can be understood as maximizing the signal-to-noise ratio. Model 2 was more focused in that the single HLA allele predictor variable in the fixed effect component consisted of only DQB1*06:02 and DRB1*15:01. Model 2 was also simpler in that we only accounted for two random effect variables, namely for individuals (in this case 2 and 6 levels for Peyron et al. and Diego et al., respectively) and experiments. Thus, Model 2 is a direct head-to-head comparison of the two main HLA alleles of interest. The Model 1 results are reported in Figure 1A and B, where it can be seen that relative to the baseline reference allele provided by DQB1*02:01, both DQB1*06:02 and DRB1*15:01 are at significantly increased risk of contributing to the overall peptide count. Results are reported as risk ratios (RRs) and their associated 95% confidence interval lower and upper bounds (95% CI LB and 95% CI UB). For DQB1*06:02 and DRB1*15:01 respectively, we found a RR (95% CI LB, 95% CI UB) of 1.76 (1.24, 2.50) and 14.16 (10.38, 19.33). Because they are compared to the same baseline, the two RRs may also be directly compared, thus showing that DRB1*15:01 contributes significantly more to the overall peptide count than DQB1*06:02. Under Model 2 (the head-to-head comparison), the RR for the DRB1*15:01 allele against the baseline DQB1*06:02 allele is 7.00 (5.80, 8.44) (Figure 2A and B). These results contribute to the field of precision medicine because they demonstrate that even in the case of alleles in tight LD, their effects can be effectively disentangled by a DC-PPPA and a log-linear mixed model analysis. Our results support the conclusion that in regard to the well-known risk haplotype of DQB1*06:02/DRB1*15:01, the true HLA risk allele seems to be DRB1*15:01. Disclosures Luu: Haplogenics Corporation: Current Employment. Hofmann:CSL Behring: Current Employment. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Dinh:Haplogenics Corporation: Current Employment. Escobar:National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maraskovsky:CSL Behring: Current Employment. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.

Factor VIII inhibitor development in Egyptian hemophilia patients: does intron 22 inversion mutation play a role?

BackgroundHemophilia A (HA) is an X-linked recessive bleeding disorder characterized by qualitative and quantitative deficiency of factor VIII (FVIII). The development of inhibitor antibodies against FVIII is the most challenging complication of treatment. Mutations in the FVIII gene is one of the genetic factors that leads to development of FVIII inhibitors especially intron 22 inversion (Inv22).ObjectivesThis study was carried out to assess the frequency of Inv22 of FVIII gene in Egyptian patients with hemophilia A and its role as a risk factor for developing inhibitors.Patients and methodsSeventy-two patients with severe HA and 48 patients with moderate HA were enrolled in the current study. All patients were treated on demand with either plasma-derived factor VIII or recombinant factor VIII concentrates. Genotyping of FVIII Inv22 was performed by LD-PCR while the presence and magnitude of inhibitor activity in blood was determined by the Bethesda assay.ResultsAround 23% of all hemophilia cases had positive Inv22. Intron 22 inversion mutation was detected in 6 and 33% of patients with moderate and severe HA respectively. Twenty-one cases (18%) of all hemophilic patients developed inhibitors. Thirty-7% of patients with Inv22 had inhibitor in their blood, almost all, but one, had severe HA. The risk of an inhibitor development during replacement therapy was four folds higher among Inv22 positive cases as compared with mutation negative peers (OR 4.3, 95% CI 1.6–11.9, P = 0.003).ConclusionsThe prevalence of Inv22 of F VIII in Egyptian hemophiliacs is nearly like that of other population. This mutation was more frequently detected among severe hemophilic patients as compared with moderately affected peers. The presence of Inv22 mutation significantly predispose to FVIII inhibitor development.

Inhibitors-Recent insights.

The development of inhibitory antibodies to therapeutic factor VIII (FVIII) in haemophilia A (HA) patients is the major complication in treatment/prevention of haemorrhages. The reasons some HA patients develop inhibitors while others do not remain unclear. This review briefly summarizes our understanding of anti-FVIII immune responses, the roles of T cells, both effector and regulatory, and generally discusses the interplay between FVIII and the immune system, both in factor replacement therapy and gene therapy, with some comparisons to factor IX and haemophilia B therapies. Notably, we propose that the prevailing observed active tolerance to FVIII in both HA and non-HA individuals rests to greater or lesser extents on peripherally induced immune tolerance. We also propose that the immune systems of inhibitor-negative HA patients do not merely ignore therapeutic FVIII, but rather have immunologically assessed and actively tolerized the patients to exogenous FVIII. Induction of such peripheral immune tolerance may further be triggered in HA patients who failed to tolerize upon initial FVIII exposure by 'appropriate' stimulation of their immune system, eg by immune tolerance induction therapy via intensive FVIII therapy, by oral administration of FVIII, by cellular therapies or by gene therapy directed to immuno-tolerogenic sites such as the liver.

B Cell Depletion Eliminates FVIII Memory B Cells and Enhances AAV8-coF8 Immune Tolerance Induction When Combined With Rapamycin.

Hemophilia A is an inherited coagulation disorder resulting in the loss of functional clotting factor VIII (FVIII). Presently, the most effective treatment is prophylactic protein replacement therapy. However, this requires frequent life-long intravenous infusions of plasma derived or recombinant clotting factors and is not a cure. A major complication is the development of inhibitory antibodies that nullify the replacement factor. Immune tolerance induction (ITI) therapy to reverse inhibitors can last from months to years, requires daily or every other day infusions of supraphysiological levels of FVIII and is effective in only up to 70% of hemophilia A patients. Preclinical and recent clinical studies have shown that gene replacement therapy with AAV vectors can effectively cure hemophilia A patients. However, it is unclear how hemophilia patients with high risk inhibitor F8 mutations or with established inhibitors will respond to gene therapy, as these patients have been excluded from ongoing clinical trials. AAV8-coF8 gene transfer in naïve BALB/c-F8e16−/Y mice (BALB/c-HA) results in anti-FVIII IgG1 inhibitors following gene transfer, which can be prevented by transient immune modulation with anti-mCD20 (18B12) and oral rapamycin. We investigated if we could improve ITI in inhibitor positive mice by combining anti-mCD20 and rapamycin with AAV8-coF8 gene therapy. Our hypothesis was that continuous expression of FVIII protein from gene transfer compared to transient FVIII from weekly protein therapy, would enhance regulatory T cell induction and promote deletion of FVIII reactive B cells, following reconstitution. Mice that received anti-CD20 had a sharp decline in inhibitors, which corresponded to FVIII memory B (Bmem) cell deletion. Importantly, only mice receiving both anti-mCD20 and rapamycin failed to increase inhibitors following rechallenge with intravenous FVIII protein therapy. Our data show that B and T cell immune modulation complements AAV8-coF8 gene therapy in naïve and inhibitor positive hemophilia A mice and suggest that such protocols should be considered for AAV gene therapy in high risk or inhibitor positive hemophilia patients.

Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice

AbstractFactor VIII (FVIII) replacement therapy for hemophilia A is complicated by development of inhibitory antibodies (inhibitors) in ∼30% of patients. Because endothelial cells (ECs) are the primary physiologic expression site, we probed the therapeutic potential of genetically restoring FVIII expression selectively in ECs in hemophilia A mice (FVIIInull). Expression of FVIII was driven by the Tie2 promoter in the context of lentivirus (LV)-mediated in situ transduction (T2F8LV) or embryonic stem cell–mediated transgenesis (T2F8Tg). Both endothelial expression approaches were associated with a strikingly robust immune response. Following in situ T2F8LV-mediated EC transduction, all FVIIInull mice developed inhibitors but had no detectable plasma FVIII. In the transgenic approach, the T2F8Tg mice had normalized plasma FVIII levels, but showed strong sensitivity to developing an FVIII immune response upon FVIII immunization. A single injection of FVIII with incomplete Freund adjuvant led to high titers of inhibitors and reduction of plasma FVIII to undetectable levels. Because ECs are putative major histocompatibility complex class II (MHCII)-expressing nonhematopoietic, “semiprofessional” antigen-presenting cells (APCs), we asked whether they might directly influence the FVIII immune responses. Imaging and flow cytometric studies confirmed that both murine and human ECs express MHCII and efficiently bind and take up FVIII protein in vitro. Moreover, microvascular ECs preconditioned ex vivo with inflammatory cytokines could functionally present exogenously taken-up FVIII to previously primed CD4+/CXCR5+ T follicular helper (Tfh) cells to drive FVIII-specific proliferation. Our results show an unanticipated immunogenicity of EC-expressed FVIII and suggest a context-dependent role for ECs in the regulation of inhibitors as auxiliary APCs for Tfh cells.

Factors Involved in the Development of Inhibitory Antibodies in Patients with Hemophilia in Colombia: A Case-Control Study.

Background:The appearance of inhibitory antibodies against antihemophilic factors is one of the most serious complications related to hemophilia.Objective:The objective of this study was to identify variables and factors related to the development of inhibitory antibodies in a group of patients undergoing antihemophilic therapy in Colombia.Methods:A case-control study in patients with hemophilia treated in Specialized Healthcare Provider Institutions (IPS-E) in 21 cities of Colombia of any age and with a diagnosis of inhibitory antibodies against factor VIII or IX during 2016. Four controls per case paired by age and type of hemophilia were used. Sociodemographic, clinical, and pharmacological variables were identified and analyzed.Results:Seventeen patients with inhibitory antibodies and 68 controls with hemophilia were identified. The mean age was 28.3 ± 17.8 years. A total of 94.1% had hemophilia A, and 88.2% of the cases and 50.0% of the controls had severe hemophilia; 47.1% of the cases and 54.4% of the controls were receiving prophylaxis with coagulation factors. Multivariate analysis showed that having severe hemophilia (OR:17.0, 95%CI:1.32–219.60) and lack of knowledge of the coagulation factor with which the patient was treated before entering the care program in the IPS-E (OR:8.9, 95%CI:1.82–43.75) were significantly associated with a higher probability of developing inhibitory antibodies.Conclusion and relevance:Coagulation factors associated with the development of inhibitory antibodies were severe hemophilia and lack of knowledge of the type of factor used prior to entering the follow-up cohort.

The national blueprint for 21st century data and specimen collection and observational cohort studies: NHLBI State of the Science Workshop on factor VIII inhibitors.

A devastating complication of hemophilia A, the congenital deficiency of coagulation factor VIII (FVIII), therapy is the development of inhibitory antibodies (inhibitors) to infused FVIII concentrate affecting up to 30% of people with the most severe form of the disease. Although the number of patients affected by hemophilia A with inhibitors is small, the physical, emotional, financial, and public health impact is overwhelming to the patient, family, and medical system. To best serve this patient population, as well as enhance knowledge around this complication, a robust data and specimen collection strategy must be designed. Working Group 2 (WG2) of the National Heart, Lung, and Blood Institute (NHLBI)- sponsored State of the Science (SOS) workshop on factor VIII inhibitors in 2018 was tasked with developing recommendations around the conduct of research, modeling efficient data and specimen collection, developing infrastructure support, establishing partnerships with all stakeholders, including international collaborators, addressing ethical issues, maximizing patient/family engagement and promoting training opportunities. A group with diverse expertise was assembled who reviewed the current state of data and specimen collection in patients with hemophilia and developed recommendations for the future. Our results were presented at the SOS Workshop where additional feedback was gained. Our charge and recommendations are summarized in this manuscript.

Reprogrammed CD4+ T Cells That Express FoxP3+ Control Inhibitory Antibody Formation in Hemophilia A Mice.

Coagulation Factor VIII (FVIII) replacement therapy in hemophilia A patients is complicated by the development of inhibitory antibodies, which often render the treatment ineffective. Previous studies demonstrated a strong correlation between induction of regulatory T cells (Treg) and tolerance to the therapeutic protein. We, therefore, set out to evaluate whether the adoptive transfer of FVIII-specific CD4+ Treg cells prevents inhibitor response to FVIII protein therapy. To this end, we first retrovirally transduced FoxP3+ into FVIII-specific CD4+ cells, which resulted in cells that stably express FoxP3, are phenotypically similar to peripherally induced Tregs and are antigen specific suppressors, as judged by in vitro assays. Upon transfer of the FVIII-specific CD4+ FoxP3+ cells into hemophilia A mice, development of inhibitory antibodies in response to administering FVIII protein was completely suppressed. Suppression was extended for 2 months, even after transferred cells were no longer detectable in the secondary lymphoid organs of recipient animals. Upon co-transfer of FoxP3+-transduced cells with the B cell depleting anti-CD20 into mice with pre-existing inhibitory antibodies to FVIII, the escalation of inhibitory antibody titers in response to subsequent FVIII protein therapy was dramatically reduced. We conclude that reprogramed FoxP3 expressing cells are capable of inducing the in vivo conversion of endogenous FVIII peripheral Tregs, which results in sustained suppression of FVIII inhibitors caused by replacement therapy in recipient hemophilia A animals.