Effect of HLA DR epitope de-immunization of Factor VIII in vitro and in vivo

Abstract

T cell-dependent development of anti-factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle to replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce HLA binding of epitopes based on predicted anchoring residues. Here, we used immunoinformatics tools to identify C2 domain HLA DR epitopes and predict site-specific mutations that reduce immunogenicity. Epitope peptides corresponding to original and modified sequences were validated in HLA binding assays and in immunizations of hemophilic E16 mice, DR3 and DR4 mice and DR3xE16 mice. Consistent with immunoinformatics predictions, original epitopes are immunogenic. Immunization with selected modified sequences lowered immunogenicity for particular peptides and revealed residual immunogenicity of incompletely de-immunized modified peptides. The stepwise approach to reduce protein immunogenicity by epitope modification illustrated here is being used to design and produce a functional full-length modified FVIII for clinical use.

Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based onPseudomonasexotoxin A

“…Evaluating T-cell activation with peptides alone is useful for showing that a known epitope is abolished (20), but it is insufficient for demonstrating that new epitopes have not been created. For instance, mutations could alter antigen processing of the protein and potentially generate new epitopes.…”

“…CD4 + T-cell support is initiated by antigen-presenting cells (APCs), which display peptide fragments derived from foreign proteins on MHC class II molecules that bind T-cell receptors (14,15). Several studies have identified human-specific T-cell epitopes in therapeutic proteins (16)(17)(18), and in some cases proteins were produced by mutating amino acids within the protein and were shown to be less immunogenic using mouse models (19)(20)(21)(22).…”

Clotting factor concentrate switching and inhibitor development in hemophilia A

“…First, the lower the intrinsic immunogenicity of a second FVIII product, the greater the probability of not breaking tolerance to epitopes shared between products. 40 Second, the host could be conditioned to mount a tolerogenic response on receiving the new product in the form of a negative vaccination strategy. 41 From a theoretical, immunologic point of view, the claim of safety issues on switching from a plasma-derived product to a molecularly defined bioengineered one, or on changing biodrug, appears to be weak.…”

T cells from hemophilia A subjects recognize the same HLA-restricted FVIII epitope with a narrow TCR repertoire

“…23 Moise et al used computational prediction, HLA-DR peptide binding assays, and immunizations of HLA-DRA*01-DRB1*03:01 and -DRB1*04:01 transgenic mice to identify 6 immunogenic peptides in the FVIII-C2 domain. 24 Van Haren et al investigated naturally processed FVIII peptides by sequencing peptides eluted from HLA-DR on dendritic cells isolated from HLA-DRB1-typed donors. 25,26 These studies identified 6 to 18 FVIII core peptides per individual that could potentially be recognized by their T cells.…”