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Thank you for attending THSNA 2026. The virtual meeting is now closed.
Thank you for attending THSNA 2026. The virtual meeting is now closed.
Presentation Details
| Factor VIII Protein Modifications Influence the Immune Recognition of FVIIIFc-VWF-XTEN in Hemophilia A Patient Samples (THSNA �Travel Awardee) Ana L.Altamiranda, Mengjie J.Kong, Huong Chau, May Chien, Elizabeth S.York, Glaivy Batsuli. Stanford University, Palo Alto, CA, USA |
Abstract
Background: FVIIIFc-VWF-XTEN is an ultra-extended half-life factor VIII (FVIII) protein containing an IgG1 Fc region, XTEN polypeptide, and the D’D3 domain of von Willebrand factor (VWF). VWF reduces FVIII uptake by dendritic cells and contributes to a reduced risk of inhibitor formation in previously untreated patients (PUPs). Thus, the inclusion of the D’D3 region may confer immunoprotective properties. We have previously shown that FVIIIFc-VWF-XTEN modifications significantly reduces binding of some affinity-matured anti-FVIII monoclonal antibodies (MAbs) in vitro. Whether this effect translates to reduce FVIII binding by inhibitor patient plasmas is unclear. Objectives: To assess the effect of FVIII protein modifications on antibody binding in hemophilia A patient samples with inhibitors. Methods: A competition ELISA was utilized to compare anti-FVIII MAbs versus pediatric & adult patient samples binding to 4 FVIII proteins: FVIIIFc-VWF-XTEN, B domain deleted FVIII (BDD FVIII), full-length FVIII (FL FVIII), and FVIII with Fc conjugation (FVIII-Fc). A subset of 5 high-affinity anti-FVIII MAbs were purified from hybridoma cell lines to perform these studies, specifically MAbs 2-116 (A1 domain), 4A4 (A2 domain), 2-113 (A3 domain), 2A9 (C1 domain), 3D12 (C2 domain). Confirmation of anti-FVIII IgG status in patient samples were confirmed by ELISA using FL FVIII. MAb binding to FVIII deficient plasma without inhibitors tested on 3 separate days from George King Biomedical, Inc was used as the control plasma. An ELISA titer ratio of test plasma (i.e., patient plasma) to control plasma was calculated and titer ratios less than 2 standard deviations of the mean of the control plasma binding to each MAb was considered significant. Results: To date, 12 hemophilia A plasma samples have been tested: 3 with active inhibitors, 3 with successfully tolerized inhibitors, 3 without any inhibitors, and 3 with acquired inhibitors. Of the 6 samples with reported inhibitors, only 4 samples (1 active inhibitor and 3 acquired inhibitors) had detectable anti-FVIII IgG ELISA titers between 129 and >5,000 at the time of sample collection. All 5 MAbs had significantly reduced binding to FVIIIFc-VWF-XTEN in the presence of active or acquired inhibitor plasma samples. In comparison, 80%, 60%, and 60% of the 5 MAbs demonstrated significantly reduced binding to BDD FVIII, FL FVIII, and FVIII-Fc, respectively, in the presence of inhibitor plasmas. Distinctively, only FVIIIFc-VWF-XTEN consistently exhibited reduced binding to MAbs targeting heavy-chain epitopes (A1/A2). This suggests increased competition for binding to FVIIIFc-VWF-XTEN by inhibitor plasmas, with pronounced effects at the FVIII heavy-chain. This also confirms recognition of the 5 main FVIII domains by inhibitor plasmas despite FVIIIFc-VWF-XTEN protein modifications. Interestingly, 3 non-inhibitor samples (2 successfully tolerized and 1 without inhibitors) demonstrated reduced binding by the 5 MAbs to FVIIIFc-VWF-XTEN followed by FVIII-Fc, which may indicate a protective effect associated with the IgG1 Fc region. Conclusions: FVIII protein modifications differentially alter binding of affinity-matured FVIII antibodies in vitro, both in inhibitor and non-inhibitor samples. Whether the observed changes in antibody binding with FVIIIFc-VWF-XTEN would translate to more rapid immune tolerance induction in inhibitor patients or reduced antibody formation in PUPs warrants further investigation.
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No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.