OC 47.5 - The impact of N-linked glycosylation in C1 domain of FVIII on immunogenicity

Monday, July 11, 2022

3:45 PM – 4:00 PM

Location: ExCel Center, ICC Capital Suite Room 2-4

Presentation Format: Virtual

Presenting Author(s)

MF

Meng-Ni Fan

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

Co-Author(s)

SW

Shuaishuai Wang

Postdoctoral Research Associate
Georgia State University
Atlanta, Georgia, United States
JZ

Junping Zhang

Research Associate
Indiana University
Indianapolis, Indiana, United States
XC

Xiaohe Cai

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

Ting-Yen Chao

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

Lei Li

Principle Investigator
Georgia State University
Atlanta, Georgia, United States
WX

Weidong Xiao

Principal Investigator
Indiana University
Indianapolis, Indiana, United States
Barbara A. Konkle, MD

Professor of Medicine
University of Washington
Seattle, Washington, United States
CM

Carol Miao

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

Background: We previously demonstrated that lower FVIII inhibitor titers were detected in hemophilia A (HemA) mice injected with FVIII plasmid containing Glutamine to Asparagine substitution in C1 domain (N2118Q) than the mice with BDD-FVIII plasmid via hydrodynamic gene delivery. It suggested that the elimination of N-glycosylation in C1 domain reduced the immunogenicity and a glycoepitope may exist in C1 domain.

Aims: To examine the impact of FVIII glycosylation following AAV-mediated gene therapy and further characterize the important immunogenic glycoepitope region in C1 domain.

Methods: HemA mice were injected with AAV-FVIII and AAV-N2118Q, followed by challenge with repeated FVIII injections. FVIII activity and inhibitor titer were examined over time. To characterize the immunogenic region around 2118 site, three sets of 15-amino-acid peptides were designed with partly overlapping and with or without mannosylation (MP1/NGP1; MP2/NGP2; MP3/NGP3). Cell proliferation and cytokine levels in supernatant were evaluated from mouse splenocytes or human PBMCs cultured with different peptides. Immunization studies in HemA mice using these peptides and FVIII challenge are being carried out.

Results: FVIII expression was initially detected in all AAV treated mice for eight weeks, however, subsequently dropped to very low or undetectable at week 12 in BDD group, whereas was only slightly decreased in N2118Q group. Following FVIII challenge, N2118Q group showed higher resistance to inhibitor formation than BDD group.
Higher proliferation rates or cytokine levels were detected in mouse cells and human PBMCs isolated from inhibitor subjects cultured with two mannosylated peptides (MP1 and MP2) but not with MP3 and all non-mannosylated peptides. Cells from control non-inhibitor subjects did not exhibit increased proliferation when stimulated with all peptides.

Conclusion(s): Elimination of N-glycosylation in C1 domain reduced FVIII immunogenicity following both plasmid- and AAV-mediated gene therapy. The glycoepitope surrounding N2118 in FVIII was identified and characterized in both human and mouse cells.