Mass spectroscopy-defined neoepitopes are a rich source of tumor rejection-mediating neoepitopes in a mouse sarcoma

Abstract

Using tandem mass spectroscopy (MS), we identified 3646 unique sequences among peptides eluted from purified K^d and D^d MHC I molecules of the BALB/c fibrosarcoma Meth A. These peptides were cross-referenced with the output of neoepitopes predicted for this tumor by our prediction pipeline CCCP (Cross Consensus Calling Platform). Eleven (11) of the eluted peptides were identified as neoepitopes and eight neoepitopes (of 11) were confirmed by targeted MS.

Each neoepitope was used to immunize BALB/c mice (twice, one week apart, using precise neoepitopes along with bone marrow-derived dendritic cells); mice were challenged with Meth A cells one week after the last immunization, and tumor growth was monitored in individual mice. In parallel, immunized mice were tested for CD8+ T cells to the neoepitopes using tetramer staining and interferon g secretion by CD8 cells.

Four of the eight neoepitopes elicited rejection of Meth A fibrosarcoma; two of the four neoepitopes elicited highly potent tumor rejection, while the other two elicited statistically significant but weaker tumor rejection. Of the two strong neoepitopes, only one elicited a measurable CD8 response. Both weak neoepitopes elicited measurable CD8 responses. Of the four neoepitopes that did not elicit tumor rejection, only one elicited a measurable CD8 response; this CD8 response was the strongest of all CD8 responses detected.

These observations indicate that MS-defined neoepitopes can be a rich source of neoepitopes that can mediate tumor rejection. Further, they highlight the fact that CD8 responses are not a good predictive surrogates for tumor rejection.