ABSTRACT: Despite increasing efficacy for standard of care of ovarian cancer (OvCa), the overall five-year survival of OvCa patients is only 47.6%. Polyclonal T cell responses against multiple cancer- and patient- specific antigens are the key element of effective cancer immunity and resulting outcomes. Project 3 develops and tests a new immunization strategy to enhance the induction of cytotoxic lymphocytes (CTLs) against multiple patient-specific epitopes, by targeting cancer cells, endogenous dendritic cells (DCs) and ex vivo generated DCs. We will compare the immunopeptidomes on OvCa cells and two populations of DC specialized in CTL induction: ex-vivo generated alpha-type-1-polarized (αDC1s) and endogenous conventional DCs (cDC1s) which share the inflammatory BATF3/IRF8 phenotype and elevated ability to cross-present multiple cancer-cell-associated antigens to CD8+ T cells. Combining our unique in vitro sensitization (IVS) and bioinformatics approaches, we will test the overall hypothesis that the mismatch between immunopeptidomes of OvCa cells and DCs presenting antigens from cancer cells limits the therapeutic effectiveness of spontaneous and vaccination-induced CTL responses. We further hypothesize that αDC1s loaded with synthetic patient-specific neoantigen peptides will bypass such mismatch, inducing CTLs particularly effective in killing OvCa tumors. We propose the following three Aims: Specific Aim 1: Compare the antigenic specificity of human CD8+ T cells induced by DCs loaded with autologous OvCa cells, tumor-eluted peptides and patient-specific neoantigen peptides identified by in-silico approaches. We hypothesize that CTLs induced by autologous cancer cell-loaded DC1s or cDC1s contain large number of CTLs which are irrelevant for tumor recognition, which deficit can be corrected by loading DCs with synthetic neoantigen peptides specific to each patient's OvCa cells. Specific Aim 2: Evaluate the immunopeptidome differences between OvCa cells and tumor-loaded αDC1s and endogenous cDC1s and test the feasibility of their adjustment. We hypothesize that immune adjuvants and inflammatory mediators can be used to modulate APM patterns and immunopeptidomes of DCs and OvCa cells, to enhance the antigenic match between arising CTLs and autologous OvCa cells. Specific Aim 3: Determine the feasibility, safety and clinical efficacy of αDC1 vaccines loaded with patient-specific neoantigen peptides combined with PD-1 blockade. Each patient will receive 8 courses of DC1 vaccines loaded with patient-specific neoantigens and pembrolizumab. Guided by the results of Aim 2, the patients may also receive systemic immune modulation to increase the visibility of their own OvCa cells to DC1-induced CTLs and reduce immune suppression. Clinical efficacy will be monitored by iORR. Predicted Impact: Results of Project 3 will be translated into development of novel therapeutic vaccines and prospectively into new modes of immune checkpoint inhibition and ado...