-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

1228 Incorporation of O-Glycopeptides into FVIII Decreases Its Immunogenicity

Program: Oral and Poster Abstracts
Type: Oral
Session: 321. Coagulation and Fibrinolysis: Basic and Translational: Coagulation Protein: Molecular Insights and Molecular Therapies
Hematology Disease Topics & Pathways:
Research, Bleeding and Clotting, Translational Research, hemophilia, Diseases, immunology, Biological Processes, molecular biology
Saturday, December 9, 2023: 9:45 AM

Meng-Ni Fan, PhD1*, Matthew Chrzanowski, PhD2*, Amber Vander Kooi1*, Tangliang Shen3*, Rodney M. Camire, PhD4, Xiaohe Cai1*, Ting-Yen Chao1*, Junping Zhang, PhD2*, Barbara A. Konkle, MD5,6, Carol H. Miao, PhD1,5, Lei Li, PhD3* and Weidong Xiao, PhD2*

1Seattle Children's Research Institute, Seattle, WA
2Indiana University, Bloomington, IN
3Georgia State University, Atlanta, GA
4The Children's Hospital of Philadelphia, Philadelphia, PA
5University of Washington, Seattle, WA
6Washington Center For Bleeding Disorders, Seattle, WA


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.


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.


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.


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.

Disclosures: Konkle: Regeneron: Consultancy; Octapharma: Consultancy; Spark: Consultancy; Sigilon: Consultancy; Pfizer: Consultancy; uniQure: Research Funding; Takeda: Research Funding; Spark: Research Funding; Pfizer: Research Funding; BioMarin: Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH