Non-Natural and Photo-Reactive Amino Acids as Biochemical Probes of Immune Function

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.

Altered Peptide Ligands Revisited: Vaccine Design through Chemically Modified HLA-A2–Restricted T Cell Epitopes

“…MHC molecules were diluted in PBS supplemented with 0.5 mg/ml b-g-globulin (Sigma-Aldrich) to a final concentration of 0.75 mM and pipetted into a 96-well microplate. Tracer peptide FLPSDCFPSV (based on FLPSDFFPSV, Hepatitis B virus core protein (18)(19)(20)(21)(22)(23)(24)(25)(26)(27) ) with a fluorescent TAMRA molecule covalently bound to the cysteine residue through a maleimide linkage was used as the competitor peptide (38). This tracer peptide was diluted in PBS/b-g-globulin to a concentration of 6 nM and pipetted into a 96-well microplate.…”

“…Epitopes modified based on amino acid substitutions are termed "altered peptide ligands" (APLs) (11). A well-known example of such an APL is the alanine to leucine modification in the melanoma-associated Mart-1/Melan-A (26)(27)(28)(29)(30)(31)(32)(33)(34)(35) epitope EAAGIGILTV that leads to enhanced MHC-binding (12).…”

“…Strategies that have been pursued to improve and stabilize MHC epitopes include the incorporation of residues such as nonencoded a-amino acids (23)(24)(25), photoreactive cross-linking amino acids (26), N-methylated amino acids (27) and b-amino acids (27)(28)(29)(30), backbone reduction (reviewed in Ref. 31), (partial) retroinversion by using D-amino acids (32,33), N-terminal methylation and C-terminal amidation (27,34) and pegylation (reviewed in Ref.…”

Fluorine substitutions in an antigenic peptide selectively modulate T-cell receptor binding in a minimally perturbing manner

“…Fluorine substitutions are often used in medicinal chemistry to enhance ligand binding affinities [6], and have recently been used to probe immune recognition [7]. We reasoned that by combining selective peptide fluorination with insight from crystallographic structures, we could target structural properties in interfaces between TCRs and their pMHC ligands, simultaneously carrying out both positive and negative design.…”

Modulating Cocaine Vaccine Potency through Hapten Fluorination

“…6 Incorporation of fluorine atoms or fluorine-containing substituents is often used to enhance ligand-binding affinities, and has recently been found to enhance immune recognition. 7 Indeed, immune response in part is based on the T cell receptor (TCR) recognition of antigenic molecules bound and presented by major histocompatibility complex (MHC). A weak interaction of TCRs with antigen-MHC may fail to evoke a significant immune response.…”