Identification of Viral and Cancer epitopes using peptide:MHC Stability measurements

The socio-economic threat from pandemics, the most recent example being SARS-CoV-2, as well as the high incidence of cancer in developed countries emphasizes the necessity of having effective anti-viral and anti-cancer therapies. Immune-targeting therapeutics, such as anti-viral vaccines and cancer immunotherapies, are important tools in this aspect. A commonly used way of identifying targets for vaccines or immunotherapy is to assess the affinity of the epitope:HLA complex. The premise being that epitopes with a high binding affinity for HLA molecules are immunogenic. Studies have however showed that the stability of the epitope:HLA complex is a stronger indicator of immunogenicity than affinity alone. The aim of this thesis was to 1) investigate the impact of epitope:HLA complex stability on the immunogenicity of SARS-CoV-2 epitopes and 2) utilize this knowledge on stability and immunogenicity to select an epitope for TCR-like antibodies as a next generation cancer immunotherapy.

Here we show that accounting for the stability of epitope:HLA complexes enriches the identification of immunogenic epitopes. This was evident by the induction of strong CD8⁺ T cell activation in response to stimulation with high stability peptides. These responses were observed for four different HLA-alleles in both SARS-CoV-2 vaccinated and convalescent donors. Deconvolution of the response revealed that the peptides with the highest stability and affinity to A*0101, A*0201 and A*0301 were inducing the response. This was confirmed by tetramer staining of antigen specific CD8⁺ T cells.

The impact of stability in the process of epitope discovery was further elucidated by identifying a novel epitope derived from the cancer testis antigen PRAME. This HLA-A*0201-binding PRAME epitope was discovered using complex-stability assay and the immunogenicity of the epitope was demonstrated by expansion of epitope-specific CD8⁺ T cells. TCR-LAs were identified using yeast and phage display and generated as scFv-Fc fusion IgGs. The TCR-LAs displayed high affinity and binding specificity to epitope:HLA-A*0201 complex and were shown to induce NK cell-mediated cytotoxicity of HLA-A2⁺ tumor cells presenting the target epitope.

Together, the data presented in this thesis demonstrates that the combination of HLA- affinity and HLA-stability measurements is an effective way of identifying immunogenic epitopes. This strategy can be employed both to viral and cancer antigens and represents a robust tool to identify novel epitopes for immune-targeting therapeutics.