[Acquired hemophilia A and emicizumab for the treatment of bleeding: two case report and a literature review]. / Hémophilie A acquise, le traitement par emicizumab peut relayer les agents by-passant : à propos de deux cas et une revue de la littérature.

Emicizumab is a bispecific antibody that mimics the function of factor VIII (FVIII) and is indicated for prophylactic use in patients with congenital hemophilia A with or without inhibitors. Acquired hemophilia A (AHA) is a rare and severe disorder causes by autoantibodies that inhibit FVIII. In AHA, acute bleeding are managed with bypassing agents but several reports described the off-label use of emicizumab. The aim of this article is to describe two cases of AHA treated with emicizumab and a review of the scientific littérature. Reports indicate that the use of emicizumab is efficacious to treat acute bleeding with less thrombotic events thant with bypassing agents and with a reduced hospitalisation duration. Nevertheless biological monitoring is more complicated with assay interferences and a persistent circulation more than 6 months after the last injection was observed for our two patients.

Large deletions and small insertions and deletions in the factor VIII gene predict unfavorable immune tolerance induction outcome in people with severe hemophilia A and high-responding inhibitors.

INTRODUCTION: Hemophilia A is an inherited bleeding disorder caused by pathogenic variants in the factor VIII gene (F8), which leads to factor VIII (FVIII) deficiency. Immune tolerance induction (ITI) is a therapeutic approach to eradicate alloantibodies (inhibitors) against exogenous FVIII in people with inherited hemophilia A. Few studies have evaluated the role of F8 variants on ITI outcome. MATERIAL AND METHODS: We included people with severe hemophilia A (FVIII ˂ 1 international units/dL) and high-responding inhibitors (≥ 5 Bethesda units/mL lifelong) who underwent a first course of ITI. Socio-demographic, clinical and laboratory data were collected. ITI outcomes were defined as total, partial successes, and failure. Detection of intron 1 and 22 inversions was performed by polymerase-chain reaction, followed by F8 sequencing. RESULTS: We included 168 people with inherited hemophilia A and high-responding inhibitors, median age 6 years at ITI start. Intron 22 inversion was the most prevalent variant (53.6 %), followed by nonsense (16.1 %), small insertion/deletion (11.3 %), and large deletion (10.7 %). In comparison with intron 22 inversion, the odds of ITI failure were 15.5 times higher (odds ratio [OR] 15.50; 95 % confidence interval [95 % CI] 4.59-71.30) and 4.25 times higher (95 % CI, 1.53-12.3) among carriers of F8 large deletions and small insertions and deletions, respectively. CONCLUSION: F8 large deletions and small insertions/deletions predicted ITI failure after a first course of ITI in patients with severe hemophilia A and high-responding inhibitors. This is the first study to show F8 large deletions and small insertions/deletions as predictors of ITI failure.

Efficacy and safety of recombinant porcine factor VIII in Japanese patients with acquired hemophilia A.

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by autoantibodies inhibiting human factor VIII (hFVIII). This phase II/III open-label study evaluated the safety and efficacy of recombinant porcine factor VIII (rpFVIII, susoctocog alfa) in adults with AHA and severe bleeding episodes in Japan (NCT04580407). The initial rpFVIII dose was 200 U/kg, with subsequent doses based on clinical measures including plasma FVIII activity. The primary efficacy endpoint was the proportion of severe bleeding episodes with a positive response to rpFVIII therapy 24 h after treatment initiation. Five patients were eligible for, and completed, rpFVIII treatment (age group: 60s-80s; median hFVIII inhibitor: 52 BU/mL; porcine FVIII [pFVIII] inhibitor: 3/5 patients). The median (range) total dose/patient was 548.4 (198-1803) U/kg with a median 3.0 infusions/patient. All patients responded positively to rpFVIII therapy at 24 h regardless of baseline pFVIII inhibitor status. rpFVIII treatment was well tolerated with no adverse events of special interest such as thromboembolic events or de novo pFVIII inhibitors. This study supports the use of rpFVIII as a novel therapy in the clinical management of patients with AHA in Japan. rpFVIII was approved for treating bleeding episodes in adults with AHA in Japan in 2024.

Peripheral Blood Lymphocyte Subsets in Factor VIII Inhibitor-Positive Patients with Severe Hemophilia A: A Case-Control Study.

INTRODUCTION AND OBJECTIVES: The present study aimed to investigate different peripheral lymphocyte subsets in patients with severe hemophilia A (HA) and factor VIII (FVIII) inhibitor production. For this, age-matched cases of 19 FVIII inhibitor-positive (IP), 21 FVIII inhibitor-negative (IN) and 45 healthy controls were selected for study. METHODS: Flow cytometry was used to analyze the peripheral lymphocyte subsets, including T, B, natural killer (NK) and NKT cells. The T cell subsets included CD3 + CD4-CD8- [double negative T (DNT)], CD3 + CD4 + CD8+ [double-positive T (DPT)], CD3 + CD4 + CD8- and CD3 + CD4-CD8+ T cells. Pairwise comparisons of absolute lymphocyte subset values were conducted among the three groups. The cut-off value for absolute lymphocyte counts was determined using receiver operating characteristic curve analysis. RESULTS: The results demonstrated that the absolute values of DPT cells in the IN and IP groups were significantly lower than those in the healthy control group (P = 0.007). The DNT values were also lower in severe HA patients with or without inhibitor than those in healthy subjects, but these differences were not statistically significant (P = 0.053). In addition, the absolute value of CD4+ Th cells in the IP group was lower than that in the healthy controls (P = 0.013). Although not statistically significant (P = 0.064), the absolute values of NKT cells were higher in the IN group compared with the IP group, and higher in the IP group compared with the healthy control group. There were no statistically significant differences in total T, B, CD8 + and NK cells among the IN, IP and healthy control groups. The cut-off value for absolute CD4+ Th cells in the IN group was < 598/µl. CONCLUSION: The decrease in absolute values of CD4+ Th cells in severe HA patients may contribute to the establishment of infused FVIII immune tolerance. If the CD4+ Th value remains > 598/µl, clinicians should be vigilant for possible FVIII inhibitor production, especially on days prior to FVIII exposure.

Anti-mCD20 in combination with α-mCXCL13 monoclonal antibody inhibits anti-FVIII antibody development in hemophilia A mice.

Anti-factor VIII (FVIII) antibody development poses a significant challenge in hemophilia A (HA) patients receiving FVIII protein replacement therapy. There is an urgent need for novel therapeutic strategies to inhibit the production of anti-FVIII inhibitory antibodies (inhibitors) in HA. This study aimed to investigate a combination monoclonal antibody (mAb) therapy targeting CXCL13 and CD20 on the development of anti-FVIII antibodies in a HA murine model, along with the underlying mechanisms involved. Specifically, mAbs targeting mouse CD20 (18B12) with an IgG2a backbone and mouse CXCL13 (2C4) with an IgG1 backbone were synthesized. HA mice with FVIII inhibitors were established, and the results revealed that the combination therapy of anti-mCD20 with α-mCXCL13 significantly suppressed anti-FVIII antibody development and induced FVIII tolerance. Furthermore, this combination therapy led to a marked reduction of peripheral and splenic follicular helper T cells and an enhancement of regulatory T cell induction, along with sustained depletion of bone marrow and splenic plasma cells in HA mice with preexisting FVIII immunity. Thus, the concurrence of blockage of CD20 and neutralization of CXCL13 hold promise as a therapeutic strategy for HA patients with inhibitors.

Retrospective study of the clinical manifestations and efficacy of immunotherapy for patients with acquired hemophilia A.

OBJECTIVE: Raising awareness of acquired hemophilia A (AHA) and early diagnosis is critical to reduce the associated mortality rate. We aimed to characterize acquired hemophilia in Chinese patients and evaluate the effectiveness of immunotherapy. METHODS: The clinical characteristics, laboratory test data, therapeutic approaches, and outcomes of 20 patients with AHA who were admitted to Xi'an Central Hospital between January 2012 and December 2020 were retrospectively studied. RESULTS: Nine of the patients (45%) were treated by single glucocorticoid administration; three (15%) with cyclophosphamide (CP) in combination with a glucocorticoid; four individuals (20%) received a combination therapy of rituximab with CP and glucocorticoid or rituximab with CP, vincristine, and a glucocorticoid; three (15%) by injection of human immunoglobulin in combination with a glucocorticoid; and one (5%) with CP alone. Six patients (30%) achieved total remission and 11 (55%) partial remission (PR), but three (15%) did not enter remission, indicating an objective response rate of 85%. CONCLUSION: Combination therapy with rituximab or intravenous human immunoglobulin achieves superior results in some patients with AHA. Immunosuppression and the administration of coagulation factors can rapidly control the disease and are efficacious, but >50% of patients only achieved PR. These findings suggest that the complete elimination of inhibitors requires prolonged immunosuppression therapy.

Emicizumab versus immunosuppressive therapy for the management of acquired hemophilia A.

BACKGROUND: Acquired hemophilia A (AHA) is an autoimmune bleeding disorder caused by neutralizing antibodies against coagulation factor VIII. Immunosuppressive therapy (IST) is standard of care to eradicate autoantibody production and protect from further bleeding but carries a risk of severe infection and mortality in frail patients with AHA. Recently, emicizumab has been studied for its potential to reduce the need for early and aggressive IST. OBJECTIVES: To compare outcomes of 2 studies that used either IST (GTH-AH 01/2010; N = 101) or prophylaxis with emicizumab (GTH-AHA-EMI; N = 47) early after diagnosis of AHA. METHODS: Baseline characteristics were balanced by propensity score matching. Primary endpoint was the rate of clinically relevant new bleeds during the first 12 weeks; secondary endpoints were adverse events and overall survival. RESULTS: The negative binominal model-based bleeding rate was 68% lower with emicizumab as compared with IST (incident rate ratio, 0.325; 95% CI, 0.182-0.581). No difference was apparent in the overall frequency of infections (emicizumab 21%, IST 29%) during the first 12 weeks, but infections were less often fatal in emicizumab-treated patients (0%) compared with IST-treated patients (11%). Thromboembolic events occurred less often with emicizumab (2%) than with IST (7%). Overall survival after 24 weeks was better with emicizumab (90% vs 76%; hazard ratio, 0.44; 95%, CI, 0.24-0.81). CONCLUSION: Using emicizumab instead of IST in the early phase after initial diagnosis of AHA reduced bleeding and fatal infections and improved overall survival.

Structural basis for inhibition of coagulation factor VIII reveals a shared antigenic hotspot on the C1 domain.

BACKGROUND: Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody inhibitors is a clinical complication in hemophilia A patients receiving FVIII replacement therapy. LE2E9 is an anti-C1 domain inhibitor previously isolated from a mild/moderate hemophilia A patient and disrupts FVIII interactions with von Willebrand factor and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined. OBJECTIVES: To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition. METHODS: FVIII was bound to the antigen binding fragment (Fab) of NB2E9, a recombinant construct of LE2E9, and its structure was determined by cryogenic electron microscopy. RESULTS: This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting that these residues represent a shared antigenic region on the C1 domain between FVIII-/- mice and hemophilia A patients. Furthermore, the FVIII:NB2E9 structure elucidates the orientation of the LE2E9 glycan, illustrating how the glycan sterically blocks interactions between the FVIII C1 domain and the von Willebrand factor D' domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain. CONCLUSION: These results describe an antigenic "hotspot" on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity.

Emicizumab as a Promising Form of Therapy for Type A Hemophilia - A Review of Current Knowledge from Clinical Trials.

Hemophilia is a plasma bleeding disorder characterized by a deficiency of certain blood clotting factors. The most common forms of this disease, i.e., type A and type B, affect approximately 400,000 people worldwide. Without appropriate treatment ensuring the proper coagulation cascade, this disease may lead to serious disability. Minimizing patient discomfort is possible via replacement therapy, consisting of the substitution of a missing coagulation factor via intravenous administration. Frequent medication and the risk related to factor inhibitors are significant disadvantages, necessitating the improvement of current therapies or the development of novel ones. This review examines the humanized bispecific antibody Emicizumab which ensures hemostasis by mimicking the action of the coagulation factor VIII, a deficiency of which causes type A hemophilia. The paper outlines the topic and then summarizes available clinical trials on Emicizumab in type A hemophilia. Several interventional clinical trials have found Emicizumab to be effective in decreasing bleeding episodes and raising patient satisfaction among various hemophilia A populations. Current Emicizumab-related trials are forecast to be completed between 2024 and 2030, and in addition to congenital hemophilia A, the trials cover acquired hemophilia A and patients playing sports. Providing a more comprehensive understanding of Emicizumab may revolutionize the management of hemophilia type A and improve quality of life. Conclusively, Emicizumab is a gentler therapy owing to subcutaneous delivery and fewer injections, which reduces injection-site reactions and makes therapy less burdensome, ultimately decreasing hospital visits and indirect costs.

Clinical immunogenicity outcomes from GENEr8-1, a phase 3 study of valoctocogene roxaparvovec, an AAV5-vectored gene therapy for hemophilia A.

Gene transfer therapies utilizing adeno-associated virus (AAV) vectors involve a complex drug design with multiple components that may impact immunogenicity. Valoctocogene roxaparvovec is an AAV serotype 5 (AAV5)-vectored gene therapy for the treatment of hemophilia A that encodes a B-domain-deleted human factor VIII (FVIII) protein controlled by a hepatocyte-selective promoter. Following previous results from the first-in-human phase 1/2 clinical trial, we assessed AAV5-capsid- and transgene-derived FVIII-specific immune responses with 2 years of follow-up data from GENEr8-1, a phase 3, single-arm, open-label study in 134 adult men with severe hemophilia A. No FVIII inhibitors were detected following administration of valoctocogene roxaparvovec. Immune responses were predominantly directed toward the AAV5 capsid, with all participants developing durable anti-AAV5 antibodies. Cellular immune responses specific for the AAV5 capsid were detected in most participants by interferon-γ enzyme-linked immunosorbent spot assay 2 weeks following dose administration and declined or reverted to negative over the first 52 weeks. These responses were weakly correlated with alanine aminotransferase elevations and showed no association with changes in FVIII activity. FVIII-specific cellular immune responses were less frequent and more sporadic compared with those specific for AAV5 and showed no association with safety or efficacy parameters.

Inhibitor eradication and treatment for acquired hemophilia A.

INTRODUCTION: Acquired hemophilia A (AHA) is a rare hemorrhagic autoimmune disorder characterized by autoantibodies against coagulation factor VIII (FVIII). In approximately half of the cases AHA does not recognize any cause (idiopathic form), while in the other cases it may be triggered by autoimmune disorders, cancers, drugs, infections, or pregnancy. Besides treating the underlying disorder, specific AHA treatment includes management of bleeding, if necessary, and inhibitor eradication. AREAS COVERED: This narrative review summarizes the main epidemiological, clinical, laboratory, and therapeutic characteristics of AHA. In particular, it is focused on the current therapeutic options for the inhibitor eradication, also showing the latest findings on the innovative therapies. A literature search strategy was performed, without temporal limits, through Medline and PubMed electronic databases. EXPERT OPINION: Various first-line and second-line immunosuppressive agents are currently available for the management of AHA. Among the latter, the anti-CD20 monoclonal antibody rituximab has been the object of intense research during the last years from investigators as innovative promising eradicating therapy for AHA. Preliminary data from the studies support the use of this drug as a first-line option for newly diagnosed AHA cases.

Immunogenicity profile of rurioctocog alfa pegol in previously treated patients with severe congenital hemophilia A.

ABSTRACT: Rurioctocog alfa pegol is an extended-half-life full-length recombinant factor VIII (FVIII) bound to 20-kDa polyethylene glycol (PEG) that has been shown to be well tolerated and efficacious in the treatment and prevention of bleeding events in previously treated patients with severe hemophilia A. Here, we present a comprehensive analysis of immunogenicity data collected during 6 clinical studies of rurioctocog alfa pegol, including a total of 360 unique previously treated patients with severe hemophilia A. The analysis included treatment-emerging FVIII-neutralizing antibodies (FVIII inhibitors); preexisting and treatment-emerging antibodies binding to FVIII, PEG-FVIII, or PEG; and treatment-emerging antibodies binding to Chinese hamster ovary host cell proteins. Moreover, the potential association between the presence of these binding antibodies and adverse events (AEs) observed in patients was investigated, and the potential impact of these antibodies on the incremental recovery of rurioctocog alfa pegol in patients was analyzed. Overall, the data indicate that rurioctocog alfa pegol is not associated with any unexpected immunogenicity characteristics. Of 360 patients, 1 patient developed a transient FVIII inhibitor with a titer of 0.6 Bethesda units per mL, which was not associated with any serious AEs. Antibodies binding to FVIII, PEG-FVIII, or PEG were not detected at the time when the inhibitor was present. Moreover, 54 of 360 patients either entered the clinical studies with preexisting binding antibodies or developed these antibodies after exposure to rurioctocog alfa pegol. These antibodies were transient in most patients and did not show any causal relationship to either AEs or spontaneous bleeding episodes.

Clinical and economic burden of immune tolerance induction in entire patients with hemophilia A: Insights from a real-world Korean setting.

INTRODUCTION: The most notable challenge facing hemophilia A treatment is the development of inhibitors against factor VIII, resulting in increased clinical and socioeconomic burdens due to the need for expensive bypassing agents (BPAs). Although immune tolerance induction (ITI) is currently the primary approach for inhibiting and reducing the inhibitors, the lengthy duration of ITI necessitates the continued use of BPA to manage bleeding episodes. In this study, we aimed to obtain real-world evidence on the clinical and economic aspects and associated burdens experienced by patients with hemophilia A with inhibitors undergoing ITI in Korea. METHODS: Claims data from January 1, 2007, to December 31, 2020, were used in this study. The study cohort comprised patients with hemophilia A undergoing ITI, who were categorized into three groups: successful, failed, or continuation of ITI. We evaluated clinical and economic burdens, including monthly healthcare visits, medication costs, and total medical expenses. RESULTS: The study involved 33 cases of ITI across 32 patients. Excluding seven continuation cases where success could not be determined at the observation point, the estimated success rate of ITI was 80.8 %. The median duration of ITI for all patients was 25.7 months. While no significant disparities were noted in the ITI duration between successful and unsuccessful cases (24.51 vs. 25.66 months), substantial discrepancies were observed in the duration of BPA usage (11.10 vs. 25.66 months) and the number of prescribed BPAs (1.79 vs. 2.97). CONCLUSION: Successful ITI reduced both clinical and economic burdens, resulting in decreased monthly medication expenses and overall medical costs.

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

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

BACKGROUND: Antibodies 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. OBJECTIVES: Evaluation of an in-house Luminex bead-based assay (LumiTope) compared with a commercially available ELISA and NBA. METHODS: The 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. RESULTS: Overall, 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. CONCLUSION: LumiTope 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.

Race and ethnicity and the success of immune tolerance induction among people with severe haemophilia A in the United States.

INTRODUCTION: Immune tolerance induction (ITI) is the only treatment to eradicate inhibitors in people with severe haemophilia A with inhibitors. Since the risk of inhibitor development is greater among Black and Hispanic persons, it has been hypothesized that race and ethnicity may influence ITI success. Limited studies have evaluated this hypothesis. AIM: To examine the success of ITI according to race and ethnicity. METHODS: Participants who entered the Community Counts (CC) Registry between 2013 and 2017, were aged ≥3 years at study entry, and received ITI were included (n = 559). The proportion of participants with successful ITI was examined with adjusted prevalence ratios (aPRs) and corresponding 95% confidence intervals (95% CIs). RESULTS: Among 559 participants, 56.9%, 19.1%, 18.1% and 4.3% were Non-Hispanic (NH) White, NH Black, Hispanic and Asian, respectively, and 1.7% were coded as other or missing. Approximately 80% of Hispanic, NH Black and NH White participants had good/very good prognosis, defined as having a pre-ITI peak inhibitor of < 200 Bethesda Units per millilitre. Nearly 60% of participants (59.7%) achieved successful ITI, 20.7% and 19.5% experienced partially successful or failed ITI, respectively. Successful ITI was non-significantly lower in NH Black (54.2%; aPR = 0.95, 95% CI 0.62-1.44) and Hispanic (55.4%; aPR = 0.89, 95% CI 0.71-1.13) relative to NH White participants (62.6%). CONCLUSION: In this study, 60% of participants in the CC Registry had successful ITI, consistent with previous studies. The proportion with successful ITI was generally comparable across racial and ethnic groups with similar prognosis. These findings do not support the hypothesis that ITI response varies according to race or ethnicity.

Multicenter assessment and longitudinal study of the prevalence of antibodies and related adaptive immune responses to AAV in adult males with hemophilia.

Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multicenter epidemiologic study was to determine the prevalence and persistence of preexisting immunity against AAV2, AAV5, and AAV8, in adult participants with hemophilia A or B. Blood samples were collected at baseline and annually for ≤3 years at trial sites in Austria, France, Germany, Italy, Spain, and the United States. At baseline, AAV8, AAV2, and AAV5 neutralizing antibodies (NAbs) were present in 46.9%, 53.1%, and 53.4% of participants, respectively; these values remained stable at Years 1 and 2. Co-prevalence of NAbs to at least two serotypes and all three serotypes was present at baseline for ~40% and 38.2% of participants, respectively. For each serotype, ~10% of participants who tested negative for NAbs at baseline were seropositive at Year 1. At baseline, 38.3% of participants had detectable cell mediated immunity by ELISpot, although no correlations were observed with the humoral response. In conclusion, participants with hemophilia may have significant preexisting immunity to AAV capsids. Insights from this study may assist in understanding capsid-based immunity trends in participants considering AAV vector-based gene therapy.