PROJECT SUMMARY/ABSTRACT Patients with advanced triple-negative breast cancer (TNBC) have a poor prognosis, with a median overall survival of ~18 months with standard-of-care (SOC) chemotherapy. However, overall survival can be extended in such TNBC patients, albeit, modestly to ~25 months with a combination of SOC and immunotherapy, reflecting the rationale that the immune system is an integral player in the overall anti-cancer response. In TNBC, the immunotherapy, known as an immune checkpoint inhibitor (ICI), is a monoclonal antibody that blocks the PD- 1/PD-L1 axis in CD8+ cytotoxic T lymphocytes (CTLs) to sustain their effector function. A prerequisite for effective ICI therapy is the availability of tumor antigens (Ags) which, in turn, are recognized by the Ag-specific CTLs. We now know that a positive effect of certain chemotherapeutics on ICI efficacy is due in part to the availability of ‘neo-Ags’ recognized by CTLs. These neo-Ags are thought to derive from therapy-induced mutations or, perhaps, to non-mutated determinants generated by post-translational protein modifications induced by immunogenic cell death. However, chemotherapy can generate yet another type of non-mutated neo-Ag which is currently overlooked. Chemotherapy mediates antitumor effects because cancer cells respond differently from normal cells to DNA damage. These interventions damage not only DNA, but also RNA components of the spliceosomes. Consequently, chemotherapy can be accompanied by a transient inhibition of splicing exposed as widespread intron retention. The pattern of DNA damage-induced intron retention is recurrent and reproducible in cells treated with the same chemotherapy. Translation of mRNA transcripts harboring retained introns results in generating shared non-mutated neo-Ags encoded by intronic sequences. Differential responses to chemotherapy-induced DNA damage manifested as repression of transcription for a set of cell replication- related genes in normal cells, but not in cancer cells with p53 gene mutations, is frequently the case for TNBC. This may result in generating cancer-specific, non-mutated neo-Ags encoded by retained introns. Some of these novel intron-encoded sequences may contain immunogenic neo-epitopes that can be recognized by CTLs. Here, we hypothesize that chemotherapy treatment of p53-mutant tumor cells generates novel ‘chemo-neoepitopes’ (CNEs), which can be identified and used as immunogens to mobilize new pools of tumor-specific CD8+ T cells. Our rationale reflects the concept that the CNE peptide sequences would be completely new to the immune system and would be capable of generating robust immune responses. In this proposal, we will identify candidate CNE peptides in Aim 1, and then in Aim 2, determine their immunogenicity and the breadth of the induced CD8+ T cell repertoire. Immunizations that elicit a CD8+ T cell repertoire similar or identical to that induced by the chemotherapy paradigm will suggest a novel CNE. Our long...