An unbiased approach to defining bona fide tumor neoepitopes that mediate cancer immunity

Identification of neoepitopes that are effective in cancer therapy is a major challenge in creating cancer vaccines. Here, using an entirely unbiased approach, we have queried all possible neoepitopes in a mouse cancer model and asked which of those are effective in mediating tumor rejection, and independently, in eliciting a measurable CD8 response. This analysis uncovers a new universe of effective anticancer neoepitopes which have strikingly different properties from conventional epitopes and suggests a novel algorithm to predict them. It also reveals that our current methods of prediction discard the majority of true anticancer neoepitopes. These results from a single mouse model have been validated in another, independent mouse cancer model, and are consistent with data with human studies. T cells elicited by the active neoepitopes identified here demonstrate a stem-like early dysfunctional phenotype associated with effective responses against viruses and tumors of transgenic mice. These abundant and novel anticancer neoepitopes can be exploited for generation of personalized human cancer vaccines. Single cell RNA-seq analysis of CD8+ PD-1+ TILs from mice immunized with a TRMN and a non-TRMN Mice (n=3 per group) were immunized with un-pulsed BMDCs or BMDCs pulsed with peptides Fam171bMUT (a TRMN) or Cd9MUT (a non-TRMN) and challenged with MC38-FABF. Tumors were harvested day 25 post tumor challenge and live CD8+PD-1+ TILs isolated by FACS and sequenced by scRNA-seq (10x genomics). Approximately 4400 CD8+ PD-1+ TILs were analyzed in each library.