Disulfide Bond Engineering to Trap Peptides in the MHC Class I Binding Groove

Abstract

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells—particularly when the antigenic peptide has relatively weak affinity for the MHC.

Human Major Histocompatibility Complex (MHC) Class I Molecules with Disulfide Traps Secure Disease-related Antigenic Peptides and Exclude Competitor Peptides

“…It is important to note that incorporation of the disulfide trap into the SCT appears to further increase the kinetics of assembly and folding, likely reflecting the improved C-terminal peptide anchoring (25). Furthermore, the disulfide trap incorporated into two different HLA-I complexes forms reliably and ablates exogenous peptide binding using a highly sensitive T cell readout.…”

“…A single point mutation (R48Q) generated by site-directed mutagenesis (QuikChange II XL, Stratagene) was required to introduce the 64-3-7 epitope (28 -32) into A2. Two point mutations were made in the G280-9V.␤ 2 m.A2 SCT and the FluM1.␤ 2 m.A2 SCT to create disulfide trap forms (25): a cysteine in the second position of the first linker and a cysteine at position 84 of the A2 heavy chain. DNA sequencing confirmed the correct sequence of all constructs.…”

“…1), and the prototype SCT made from K b /OVA has been well characterized in terms of assembly and expression in cells, biochemical stability, structure, T cell recognition, and incorporation of a disulfide trap to secure peptide (10,18,25). Although there are now several reports of SCTs made with HLA-I complexes (Table 1), these SCTs have not been characterized for biochemical stability, incorporation of disulfide traps, and exclusion of exogenous peptides.…”

“…To alleviate the potential for the SCT to bind exogenous peptides, we recently engineered a disulfide trap into the OVA.m␤ 2 m.K b SCT (25). Two cysteine residues were introduced by site-directed mutagenesis creating a strategically placed disulfide bond to anchor the antigenic peptide moiety of the SCT to the peptide binding groove (Fig.…”

“…To better accommodate linker extension and also improve C-terminal peptide anchoring, we recently engineered a disulfide trap into murine SCT molecules (dtSCTs) between position 84 of the MHC-I heavy chain and the second position on the SCT linker extending from the C terminus of the peptide (24,25). Not only did the dtSCT constructs fold efficiently in cells, but the disulfide trap oxidized in a reliable manner and displayed a tenacious ability to prevent the binding of exogenous peptides.…”