CRISPR-guided reversion reveals the immunogenicity of a “non-MHC binding” cancer neoepitope in vivo

Abstract

A high- affinity MHC I-peptide interaction is considered essential for immunogenicity. However, some neoepitopes with low affinities for MHC have been reported to elicit CD8-dependent tumor rejection in immunization-challenge studies. Here, we ask if a non-binder, tumor-rejection- mediating neoepitope influences the natural immunogenicity of a tumor in vivo, in the absence of artificial immunization. A mutation in tumor MUT1 was edited to its WT counterpart; the mutation was then re-introduced into the WT tumor, recapitulating the mutation in a tumor MUT2. TILs from all three tumors show T cell activation. However, TILs of MUT1 and MUT2 show significantly stronger transcriptional signatures of cytotoxicity and TCR engagement as well as the greater breadth of TCR reactivity than those of WT. Structural modeling of the Kd-neoepitope complex suggests increased hydrophobicity of the neoepitope surface consistent with higher TCR reactivity. These results reveal the immunogenicity in vivo of low affinity or “non-binding” epitopes that do not follow the canonical view of MHC I-peptide recognition.