J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access INTRODUCTION Immune checkpoint inhibitors (ICIs) have resulted in major treatment advantages for solid cancers.1 In partic- ular, ICIs that target the PD- 1/PD- L1 axis are now the standard of care for several cancers, including advanced melanoma.2 Still, many patients do not benefit from ICI therapy.3 The tumor mutational burden (TMB) is suggested to be a predictive biomarker for treatment outcome of ICI immunotherapy,4 5 however, it is hypoth- esized to be of greater importance whether tumor mutations give rise to immunogenic MHC- presented neoantigens.6 Therefore, combining ICI with a personal- ized neoantigen cancer vaccine to elicit tumor- specific T cell responses could be an attractive therapeutic avenue to augment the effect of ICI treatments. Several clinical trials are currently evaluating person- alized cancer vaccines against neoantigens using either peptides, mRNA, or DNA to induce neoantigen- specific immune response in cancer patients.7 Results from these trials show that these treatments can induce profound neoantigen- specific immune responses in humans and have a clinical relevant effect. Noteworthy, it has been shown in resectable melanoma patients that neoantigen vaccination in combination with anti- PD- 1 lowers the risk of recurrence compared with anti- PD- 1 treatment alone.8 Hence, neoantigens are promising targets for cancer treatment and are further subject of investigations. One class of neoantigen vaccines uses synthetic long peptides to induce patients’ neoantigen- specific T- cell responses. Peptides directly prime the immune system and have the advantages of easy and fast production. Clinical trials have shown that peptide vaccines targeting neoan- tigens can effectively induce T- cell responses,9 predom- inantly reporting induction of tumor- specific CD4+T cells, while induction of neoantigen- specific CD8+T cells is observed less consistently.10 11 In these trials, different strategies for neoantigen; selection (vaccine design), delivery (vaccine modality), dose, and formulation have been applied, profoundly affecting T cell activation and function and thus clinical effect.12–14 Hence, concluding on those important parameters for peptide- based neoan- tigen vaccines is challenging based on previous studies. Peptide dose has been investigated in other non- neoepitope- based cancer vaccine trials, where dose- dependent immune responses and clinical responses (CR) were observed.14 Similar mechanisms might be true for neoantigen peptide vaccines. While multiple different bioinformatic pipelines have been developed for neoan- tigen identification15 only a minority has been applied to design vaccines in clinical trials. Collaborative efforts, such as the TESLA consortium, have furthermore demon- strated that these pipelines, in general, do not identify the same neoantigens as important for treatment.16 Hence, knowledge about the impact of selection algorithms on neoantigens, relevance, immunogenicity, and effect is limited. Therefore, further studies are needed to under - stand the potential of personalized neoantigen peptide vaccines.We have previously reported interim phase I data demonstrating feasibility of the EVX- 01 neoantigen targeting and CAF09b adjuvanted peptide vaccine.9 Here, we report the complete findings of the study, including the impact of the neoantigen peptide dose and the PIONEER neoantigen quality score on T- cell activation as well as CR in patients. Patients with metastatic melanoma were vaccinated with the neopeptide vaccine EVX- 01, at three different dose levels while keeping the CAF09b adjuvant/peptide ratio constant. To enhance CD8+T cell immune responses, the vaccine was administrated by three IP followed by three intramuscular (IM) injections (reference med IP og CD8). The patients received anti- PD- 1 therapy during vaccine production and the dosing with EVX- 01 vaccine. The CD4+ and CD8+ T cell immune responses toward vaccine neopeptides were measured and we investigated the relationship between neoantigen immune responses, vaccine doses, clinical outcomes, and PIONEER quality score for selecting neoantigens. PATIENTS, MATERIALS, AND METHODS Patients Included patients (≥18 years of age) had biopsy- verified advanced unresectable melanoma. Patients were planned to either begin first- line treatment with an anti- PD- 1 checkpoint inhibitor (cohort A) or had already been treated with an anti- PD- 1 agent for at least 4 months with stable disease (SD) and qualified for continued treatment with anti- PD- 1 (cohort B). Additional inclusion criteria were as follows: ≥1 measurable lesion as per investigator- assessed Response Evaluation Criteria in Solid Tumors (RECIST V.1.1); an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; adequate organ function; and tumor tissue available for whole- exome sequencing (WES). Main exclusion criteria included severe autoimmune disease and previous severe immune- related adverse events (AEs) and no other significant comorbidity. Trial design To assess the safety, immunogenicity, and feasibility of manufacturing the personalized neoepitope- based cancer vaccine; EVX- 01- CAF09b, we conducted a phase I first- in- human clinical trial (EudraCT No. 2018- 002892- 16 and ClinicalTrials. gov NCT03715985) at the National Center for Cancer Immune Therapy (CCIT- DK) and the Depart- ment of Oncology, Copenhagen University Hospital, Herlev, Denmark. 17 18 Patients were treated with an anti- PD- 1 agent according to local guidelines (pembrolizumab 2 mg/kg, every 3 weeks or nivolumab 6 mg/kg, every 4 weeks), and the personalized cancer vaccine EVX- 01- CAF09b was added to the treatment schedule when manufacturing was completed (figure 1A). Patients received EVX- 01 treatment every 2 weeks for six EVX- 01 vaccinations in total. The first three vaccinations were administered 3 Mørk SK, et al. J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access intraperitoneal (IP), and the last three vaccinations were administered intramuscular. The trial was amended to include three- dose- level groups.