Abstract
Wilms tumor protein (WT1) is a transcription factor selectively overexpressed in leukemias and cancers; clinical trials are underway that use altered WT1 peptide sequences as vaccines. Here we report a strategy to study peptide-MHC interactions by incorporating non-natural and photo-reactive amino acids into the sequence of WT1 peptides. Thirteen WT1 peptides sequences were synthesized with chemically modified amino acids (via fluorination and photo-reactive group additions) at MHC and T cell receptor binding positions. Certain new non-natural peptide analogs could stabilize MHC class I molecules better than the native sequences and were also able to elicit specific T-cell responses and sometimes cytotoxicity to leukemia cells. Two photo-reactive peptides, also modified with a biotin handle for pull-down studies, formed covalent interactions with MHC molecules on live cells and provided kinetic data showing the rapid clearance of the peptide-MHC complex. Despite “infinite affinity” provided by the covalent peptide bonding to the MHC, immunogenicity was not enhanced by these peptides because the peptide presentation on the surface was dominated by catabolism of the complex and only a small percentage of peptide molecules covalently bound to the MHC molecules. This study shows that non-natural amino acids can be successfully incorporated into T cell epitopes to provide novel immunological, biochemical and kinetic information.
Highlights
Specific T cell mediated immune responses involve Tlymphocytes that respond to linear peptide epitopes, typically between 8 and 20 amino acids in length
Canonical medium to high affinity anchor motif peptides for major histocompatibility complex (MHC) class I make use of long hydrophobic amino acids to bind to the MHC anchor pockets, so we hypothesized that increasing the hydrophobicity in the area of the anchor residues could induce better binding and immunogenicity [21]
In contrast with the other nonnatural amino acid sequences, which were modified in or near the position 2 anchor residue, WT1J-W4WF has the fluorinated amino acid substitution in position 4, which is not an anchor residue, because hydrophobic structural elements on central regions may play a critical role in eliciting cytolytic T lymphocytes (CTLs) responses [24]
Summary
Specific T cell mediated immune responses involve Tlymphocytes that respond to linear peptide epitopes, typically between 8 and 20 amino acids in length. The peptides recognized by CD8+ T cells are 8–10 amino acids in length and are presented by class I major histocompatibility complex (MHC) molecules on the target cells. The stability of the peptide/MHC (pMHC) complexes correlates generally with the strength of the T cell response to the epitope. Advances in the understanding of the cellular immune response to peptide antigens and structural studies of the pMHC have led to different strategies for improving cancer vaccines. One frequently studied antigen is the Wilms tumor protein (WT1), a zinc-finger transcription factor expressed during normal ontogenesis [1,2,3]. Short peptides derived from WT1 protein have been identified that generate a WT1-specific cytotoxic response [11,12,13,14,15,16]
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