Published by BMJ. ABSTRACT Background Neoantigens can serve as targets for T cell- mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide- based neoantigen vaccine EVX- 01, formulated in the liposomal adjuvant, CAF09b, was safe and able to elicit EVX- 01- specific T cell responses in patients with metastatic melanoma. Here, we present results from the dose- escalation part of the study, evaluating the feasibility, safety, efficacy, and immunogenicity of EVX- 01 in addition to anti- PD- 1 therapy. Methods Patients with metastatic melanoma on anti- PD- 1 therapy were treated in three cohorts with increasing vaccine dosages (twofold and fourfold). Tumor- derived neoantigens were selected by the AI platform PIONEER and used in personalized therapeutic cancer peptide vaccines EVX- 01. Vaccines were administered at 2- week intervals for a total of three intraperitoneal and three intramuscular injections. The study’s primary endpoint was safety and tolerability. Additional endpoints were immunological responses, survival, and objective response rates. Results Compared with the base dose level previously reported, no new vaccine- related serious adverse events were observed during dose escalation of EVX- 01 in combination with an anti- PD- 1 agent given according to local guidelines. Two patients at the third dose level (fourfold dose) developed grade 3 toxicity, most likely related to pembrolizumab. Overall, 8 out of the 12 patients had objective clinical responses (6 partial response (PR) and 2 CR), with all 4 patients at the highest dose level having a CR (1 CR, 3 PR). EVX- 01 induced peptide- specific CD4+ and/or CD8+T cell responses in all treated patients, with CD4+T cells as the dominating responses. The magnitude of immune responses measured by IFN-γ ELISpot assay correlated with individual peptide doses. A significant correlation between the PIONEER quality score and induced T cell immunogenicity was detected, while better CRs correlated with both the number of immunogenic EVX- 01 peptides and the PIONEER quality score.Conclusion Immunization with EVX- 01- CAF09b in addition to anti- PD- 1 therapy was shown to be safe and well tolerated and elicit vaccine neoantigen- specific CD4+and CD8+ T cell responses at all dose levels. In addition, objective tumor responses were observed in 67% of patients. The results encourage further assessment of the antitumor efficacy of EVX- 01 in combination with anti- PD- 1 therapy.WHAT IS ALREADY KNOWN ON THIS TOPIC ⇒Tumor mutational burden has previously been correlated to a better clinical outcome to immune checkpoint inhibitor (ICI) treatment. Several clinical trials are, therefore, aiming to boost a cancer im- mune response through personalized neoantigen vaccines. However, immunogenicity of different potential neoantigens can vary significantly and can therefore have a major impact on the antitumor response. WHAT THIS STUDY ADDS ⇒We demonstrate through a dose escalation that the neopeptide vaccine EVX- 01- CAF09b is safe at high- dose levels (2000 µg total peptide) in combination with ICI therapy, where the majority of patients showed a clinical response. ⇒The PIONEER predicted neoantigens included in the vaccine induced both CD4 and CD8 T cell responses. ⇒Clinical responses correlated with PIONEER predic- tion score, which indicates PIONEER as a promising neoantigen prediction tool. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ⇒Neopeptide- based vaccines are safe to use in high- dose levels in combination with ICI therapy. This could potentially allow administration of higher number of different neopeptide for a broader cover - age in future trials. 2 Mørk SK, et al.