PB0248 - Reduced immunogenicity of FVIII through O-Glycopeptide Integration

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Meng-Ni Fan

Research Scientist
Seattle Children's Research Institute
Seattle, Washington, United States

Co-Author(s)

MC

Matthew Chrzanowski

Fellow - PhD
Indiana University
Indianapolis, Indiana, United States
AV

Amber Vander Kooi

Research Scientist
Seattle Children's Research Institute
Seattle, Washington, United States
TS

Tangliang Shen

PhD student
Georgia State University
Atlanta, Georgia, United States
RC

Rodney Camire

Professor
The Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
XC

Xiaohe Cai

Research Scientist
Seattle Children's Research Institute
Seattle, Washington, United States
Ting-Yen Chao

Research Scientist
Seattle Children's Research Institute
Everett, Washington, United States
JZ

Junping Zhang

Fellow - PhD
Indiana University
Indianapolis, Indiana, United States
Barbara A. Konkle

Professor of Medicine
Washington Center for Bleeding Disorders and the University of Washington, Seattle, WA, USA
Seattle, Washington, United States
CM

Carol H. Miao

Principal Investigator
Seattle Children's Research Institute
Seattle, Washington, United States
LL

Lei Li

Assistant Professor
Georgia State University
Atlanta, Georgia, United States
WX

Weidong Xiao

Professor
Indiana University
Indianapolis, Indiana, United States

Background: Development of inhibitors in hemophilia A (HA) patients under infusion therapy remains a significant complication. It is hypothesized that post-translational modifications on rFVIII proteins expressed in various cell lines can contribute to differential immunogenicity of FVIII. In particular, the impact of O-glycans on FVIII inhibitor development has not been well studied despite that they are found on >80% of the mammalian surface and secreted proteins.

Aims: Investigate how additional O-linked glycosylation affects FVIII immunogenicity.

Methods: 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. The FVIII activity and immunogenicity were evaluated in HA mice via plasmid transfer and protein infusion. The FVIII proteins were expressed in BHK cells and purified for LC-MS/MS-based glycomics analysis.

Results: Significantly reduced inhibitor titers were found in mice injected with plasmid containing α-2-HS and FibA sequences than FVIII-BDD group at week 4 and week 8, despite no difference in FVIII activity. Relatively higher abundance of O-glycans throughout all domains was found in FVIII-FibA compared to the BDD-FVIII. 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 difference in in vivo activity and protein half-life.

Conclusion(s): 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.