High-Throughput Stability Screening of Neoantigen/HLA Complexes Improves Immunogenicity Predictions

Mutated peptides (neoantigens) from a patient's cancer genuine can serve as targets for T-cell immunity, but identifying which peptides can be presented by an MHC molecule and elicit I cells has been difficult. Although algorithms that predict MHC binding exist, they are not yet able to distinguish experimental differences in half-lives of the complexes (an immunologically relevant parameter, referred to here as kinetic stability). Improvement in determining actual neoantigen peptide/MHC stability could be important, as only a small fraction of peptides in most current vaccines are capable of eliciting CD8(+) T-cell-cell responses. Here, we used a rapid, nigh throughput method to experimentally determine peptide/HLA, thermal stability on a scale that will be necessary for analysis of neoantigens from thousands of patients. The method combined the use of LTV-cleavable peptide/HLA class I complexes and differential scanning fluorimetry to determine the I'm values of neoantigen complexes. Measured T-m values were accurate and reproducible and were directly proportional to the half-lives of the complexes. Analysis of known HLA-A2-restricted immunogenic peptides showed that Tor values better correlated with immunogenicity than algorithm-predicted binding affinities. We propose that temperature stability information can be used as a guide for the selection of neoantigens in cancer vaccines in order to focus attention on those mutated peptides with the highest probability of being expressed on the cell surface.