The development of inhibitory antibodies to therapeutic FVIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-FVIII immune response is FVIII interaction with receptor(s) on antigen-presenting cells followed by endocytosis and presentation to naive CD4+ T cells. Recent studies indicate a role for the C1 domain in FVIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic FVIII uptake. Co-incubation of FVIII with BO2C11, a monoclonal C2-specific IgG, reduced FVIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished FVIII immunogenicity in FVIII-deficient mice. Mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent FVIII immunogenicity in mice. BO2C11 prevents FVIII binding to von Willebrand factor, thus potentially biasing FVIII immunogenicity by perturbing its half-life. Interestingly, a FVIIIY1680C mutant, that does not bind VWF, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in FVIII-deficient mice. Co-incubation of FVIIIY1680C with BO2C11 however resulted in decreased FVIII immunogenicity in vivo. Besides, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of FVIII are implicated in immunogenic FVIII uptake, at least in vitro. Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on FVIII immunogenicity.
- Received April 27, 2016.
- Accepted October 3, 2016.
- Copyright © 2016, Ferrata Storti Foundation