PURPOSE: Personalized vaccines targeting multiple neoantigens (nAgs) are a promising strategy for eliciting a diversified antitumor T cell response to overcome tumor heterogeneity. NOUS-PEV is a vector based personalized vaccine, expressing 60 nAgs and consists of priming with a non-human Great Ape Adenoviral vector (GAd20) followed by boosts with Modified Vaccinia Ankara (MVA). Here, we report data of a phase Ib trial of NOUS-PEV in combination with pembrolizumab in treatment naïve metastatic melanoma patients (NCT04990479). EXPERIMENTAL DESIGN: The feasibility of this approach was demonstrated by producing, releasing and administering to six patients 11 out of 12 vaccines within 8 weeks from biopsy collection to GAd20 administration. RESULTS: The regimen was safe, with no treatment-related serious adverse events observed and mild vaccine-related reactions. Vaccine immunogenicity was demonstrated in all evaluable patients receiving the prime/boost regimen, with detection of robust neoantigen specific immune responses to multiple neoantigens comprising both CD4 and CD8 T cells. Expansion and diversification of vaccine-induced TCR clonotypes was observed in the post-treatment biopsies of patients with clinical response providing evidence of tumor infiltration by vaccine-induced neoantigen-specific T cell. CONCLUSIONS: These findings indicate the ability of NOUS-PEV to amplify and broaden the repertoire of tumor reactive T cells to empower a diverse, potent and durable antitumor immune response. Finally, a gene signature indicative for reduced presence of activated T cells together with very poor expression of the antigen processing machinery (APM) genes has been identified in pre-treatment biopsies as a potential biomarker of resistance to the treatment.