De-Immunization of Human Factor VIII: Identification of Epitopes in the C2 Domain

Abstract

Most immune responses to protein antigens are dependent on T-cell recognition of discrete peptide epitopes presented in an MHC groove. The pattern of peptide recognition can be predicted from the primary structure of a given protein based on residues that bind (anchor) to a given HLA phenotype. Algorithms such as EpiMatrix can be applied to identify such epitopes and measure the potential immunogenicity of proteins based on epitope content. One approach to reduce immunogenicity is to generate recombinant proteins whose constituent epitopes have been modified so as to reduce their HLA binding. Modification of the sequence can be performed in silico using algorithms such as OptiMatrix. This “de-immunization” method has been used effectively with a number of therapeutic proteins already in use in clinical trials. It may therefore also be possible to target those residues of fVIII that, while contributing to HLA binding, can be mutated without altering the functional ability of fVIII to initiate clotting. We have begun this de-immunization process with the fVIII, starting with C2 domain because C2 has been confirmed to be a major target of both T cells and inhibitory antibodies. Using EpiMatrix, we selected 10 peptides in human fVIII that would be predicted to bind to eight class II HLA DR molecules that encompass over 95% of the U.S. population. These epitopes were synthesized and eight of the ten were assayed in vitro and found to bind to HLA at IC50 <100μM. Immunization of fVIII knockout mice with whole fVIII or with constituent fVIII peptide epitopes resulted in significant T cell proliferation to the peptide epitopes as measured by thymidine incorporation assays. By contrast, homologous peptide epitopes modified by 1–2 residues elicited significantly lower levels of proliferation. These preliminary results point to the feasibility of generating a de-immunized fVIII molecule with reduced immunogenicity; validation of these results is planned. If such proteins retain even partial activity to initiate clotting, then they would become useful in treating hemophilia A patients to avoid inhibitor formation. (Supported by NIH R43 HL088834-01)