Improving peptide vaccine manufacturability without sacrificing immunogenicity: substitution of methionine and cysteine with oxidation-resistant isosteres

AO_SCPLOWBSTRACTC_SCPLOWVaccines comprising peptide antigens for inducing T cell immunity are being developed for a broad range of therapeutic applications including prevention and treatment of cancer, autoimmunity, and infectious diseases. However, many peptide antigens contain cysteine and/or methionine, which are prone to form oxidation products that can present challenges to manufacturing and reduce biological activity. To address this challenge, we introduced oxidation resistant (OXR) antigens wherein the cysteine and methionine residues of naturally occurring, wild type (WT) peptide antigens are substituted with isosteric residues that are structurally related but omit the oxidation-prone sulfur atom. Our results showed that vaccination with OXR antigens substituting cysteine and methionine with isosteres alpha-aminobutyric acid and norleucine, respectively, induced immune responses to the WT antigen that were equivalent or higher than those induced by vaccination with WT antigens. T cell responses were not affected by the position of the amino acid substitutions indicating that the isosteres do not negatively impact major histocompatibility complex (MHC) binding or T cell recognition. The T cells induced were high quality and associated with anti-tumor efficacy in vivo. Interestingly, substitution of cysteine with serine, which replaces the sulfur for an oxygen, did not yield cross-reactive T cell responses, highlighting the high degree of molecular discernment of peptide-MHC processing and presentation. In sum, OXR antigens provide a generalizable strategy for eliminating sulfur oxidation products and improving the manufacturability and shelf-life of peptide-based vaccines without affecting desired biologic activity.