Incorporation of O-Glycopeptides into FVIII Decreases Its Immunogenicity

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.1228-1228
• Main Authors: Fan, Meng-Ni, Chrzanowski, Matthew, Vander Kooi, Amber, Shen, Tangliang, Camire, Rodney M., Cai, Xiaohe, Chao, Ting-Yen, Zhang, Junping, Konkle, Barbara A., Miao, Carol H., Li, Lei, Xiao, Weidong
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-187093

Introduction For patients with hemophilia A, the generation of inhibitors (neutralizing antibodies against FVIII) during treatment remains a significant complication. Clinical studies have demonstrated that patients treated with plasma-derived FVIII (pdFVIII) have a lower incidence of inhibitor development compared to those treated with recombinant FVIII (rFVIII) products. Different post-translational modifications on rFVIII proteins expressed in various cell lines may contribute to the differences in FVIII immunogenicity. Our recent study showed that N-glycosylation affects the development of inhibitors. On the other hand, the presence, and effects of O-glycans on FVIII inhibitor development are less studied, though they are found on >80% of the mammalian surface and secreted proteins. We recently identified highly and differentially expressed O-glycans on various FVIII products. This work aims to investigate how additional O-linked glycosylation might impact the immunogenicity of recombinant FVIII. Method FVIII-α2HS and FVIII-FibA plasmids were constructed by incorporating sequences encoding 6 repeats of short peptide of α-2-HS and Fibrinogen A (FibA), respectively into B-domain deleted FVIII (BDD-FVIII) plasmid. These short peptides are known to be highly O-glycosylated. The FVIII expression and immunogenicity were evaluated by introducing the plasmids into hemophilia A mice through hydrodynamic injection. Subsequently, the FVIII-FibA and BDD-FVIII proteins were expressed in BHK cells and purified for LC-MS/MS-based glycomics analysis. We assessed the activity and half-life of FVIII proteins using an aPTT assay with mouse plasma samples collected at various time points within 96 hours after a single intravenous injection. The immunogenicity of FVIII proteins was evaluated using the Bethesda assay from plasma samples collected from hemophilia A mice after weekly intravenous injection for four weeks. Results Mice treated with plasmid containing α-2-HS and FibA sequences had lower inhibitor titers, especially mice with FVIII-FibA plasmid showing significantly lower inhibitor titers than FVIII-BDD group at week 4 and week 8 after the injection, despite no difference in FVIII activity. Glycosylation analysis revealed that the FVIII-FibA protein displayed a relatively higher abundance of O-glycans throughout all domains compared to the BDD-FVIII protein. The inserted FibA sequence itself is O-glycosylated. Similar to plasmid treatment, mice injected with FVII