Incorporation of O-Glycopeptides into FVIII Decreases Its Immunogenicity
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 
Published: December 2023

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 &gt;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 FVIII-FibA protein had significantly lower inhibitor titers (1.3±1.0 BU) than mice injected with BDD-FVIII protein (37.5±37.2 BU) and commercial rFVIII products (19.8±20.2 BU) with no observed difference in in vivo activity and protein half-life.
Conclusion
The introduction of extra O-linked glycosylated peptides between A2 and A3 domains of FVIII altered O-glycosylation pattern and substantially reduced the immunogenicity without affecting FVIII activity. Our findings underscore the significant impact of post-translational modification on protein immunogenicity, particularly in the case of FVIII. Additionally, FVIII-FibA shows promising potential for clinical applications, not only in the development of new recombinant FVIII products but also in gene therapy approaches.