Note that measurement is not completely internal independent as they are confounded based on patient response, hence caution should be taken when interpretating the p values. Significant difference is indicated with asterisks (*p<0.05, **p<0.01). (H) PIONEER quality score impact on progression- free survival (PFS). Kaplan- Meier curves were generated by grouping patients based on the median PIONEER4.0 quality score of administered neoepeptides, in two balanced groups (high and low- quality score) (I) TMB impact on PFS. Kaplan- Meier curves were generated by grouping patients based on tumor mutational burden (TBM) (balanced high/low) calculated from NGS data using FDA guidelines (https://www.accessdata.fda.gov/cdrh_docs/pdf17/P170019B.pdf). BOR, best overall response; CR, clinical response; PD, progressive disease; PR, partial response; SD, stable disease. 14 Mørk SK, et al. J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access 12 patients (66%) showed an objective response (CR and PR). Given the low number of patients and that all patients initiated anti- PD- 1 treatment prior to the addi- tion of the EVX- 01- CAF09b vaccine, it is not possible to conclude on the potential clinical benefit of adding the vaccine from this study. However, responses compare favourably to expected responses by anti- PD- 1 treat- ment alone and peptide- based neoantigen treatments in addition to ICI treatment previously reported by Ott et al (59% in 27 patients).28 Patient 8 had SD at baseline after ICI treatment and developed PR after six vaccina- tions (subcutaneous target lesion decrease 10mm to 0 mm, subcutaneous abdominal non- target lesion (biopsi) decreased from 10 mm to 0. Only sign of disease was PET positive lymph node, which could indicate that the EVX- 01- CAF09b boosted antitumor efficacy. However, further studies are needed to conclude on the clinical benefit of EVX- 01. We found robust EVX- 01- specific T- cell responses in all patients, with 58% of administered neopeptides identified as immunogenic in the IFN-γ ELISpot assay. We observed that neopeptide dose correlates with magnitude but not breadth of response. To our knowledge, this is the first study to report on a dose–response correlation between neopeptides and T- cell response. Previously, studies with non- neoantigen or personalized antigens have shown both an increase and decrease in immune response with increasing doses.30 31 As T cell response magnitude increased with peptide dose and no dose- related toxicity was observed a recom- mended phase- 2 dose was established as 200 µg/peptide, 2000 µg total peptide load. While the EVX- 01 specific T- cell responses were main- tained over time, we observe that the frequency of the T cell response decline with time in the follow- up samples (online supplemental figure 4), with the lowest response observed in the time point before the patients had disease progression. Perhaps because other cancer cell clones where then responsible for the progression of disease. Hence, future clinical trials might benefit from adminis- tration of a EVX- 01 vaccine, with newly predicted peptides, at later time points to boost the immune response. To induce a stronger CD8 T cells response, we used the adjuvant CAF09b comprising the TLR3 agonist poly(I:C).32 Additionally, patients were vaccinated through the IP route for the first three vaccination, as it has previously been described that IP administration route in mouse models elicited stronger CD8+T cell responses than subcutaneous administration.32 However, we could not confirm an increase in CD8+T cell response after IP administration (T3) compared with IM administration (T4). The trial design was not optimal for delineating the differences between IP and IM immunizations. As IP administration carries higher patient risk future designs could include a design for testing if IP administration add significant increase in T cell responses. This has to some extent been investigated using a cancer- associated antigen peptides and CAF09b in.19 Furthermore, the immunogenic responses were predominantly CD4+T cell responses, although CD8+T cell responses were observed in 7/12 patients across dose levels. These observations are similar to those from other trials reporting CD4+T cell dominated responses toward peptide- based neoantigen vaccines.10 11 28 33 CD4+T cells play a role in priming and enhancing CD8+T cells34 35 and in overcoming ICI resis- tance.36 Hence, CD4+T cell responses might augment ICI treatment substantially. Vaccine- embedded neoepitope reactive CD8+T cells detected by MHC multimer stainings (VaccNARTs) were of low frequency in expanded PBMCs. We were unable to detect VaccNARTs for most immunogenic peptides measured by ELISpot. This might be due to the wrong minimal epitope being investigated. Two vaccine- induced responses were detected in patient 6 by MHC multimer staining’s were not detected by ELISpot, which could indi- cate that responses of smaller magnitude can be detected by multimer staining. A more pronounced CD8+T cell response could potentially also be detected with ELISpot if using a short EVX- 01 peptide pool for assay stimulation. However, this was not feasible due to sample limitations. We examined CD8+T cell responses in PBMCs, to inves- tigate the effect of EVX- 01- CAF09b vaccine unbiased by prestimulation. No obvious neoepitope- spreading within non- vaccine- related predicted neopeptides was detected following the vaccination. However, one patient showed an increase in NARTs after initiation of ICI treatment, comparable to a previous study reporting an ICI- induced increase in NARTs in the blood.37 Importantly, we only had access to blood samples and not tumor samples collected after vaccination for this analysis. Ideally, it would have been beneficial to analyze TILs from tumor samples obtained postvaccination, which would have allowed us to detect potentially new vaccNARTs within the tumor microenvironment. This could better indi- cate epitope- spreading, as demonstrated in previous studies.28 We further compared the clinical outcome, BOR, to the immunogenicity of the EVX- 01 neopeptides and found that a higher fraction of, as well as, the number of immunogenic EVX- 01 peptides was correlated with CR. It appears that the number of immunogenic peptides might be important for CR and longer PFS, rather than the magnitude of response toward single vaccine- embedded peptides. A broader T- cell response will reduce the risk of tumor escape through antigen loss.6 38 Finally, we evaluated the neoantigen quality scores obtained with the AI prediction tool PIONEER used to identify EVX- 01 peptides with our immunogenicity results. The newest version of PIONEER (V.4.0) had a larger split between immunogenic and non- immunogenic based on quality scores and better placement of VaccNART- detected EVX- 01 peptides. These results, together with a better positive correlation between clinical outcome and prediction score, show that the newer version of PIONEER is better at predicting immunogenic peptides with CD4+and CD8+ T cell responses. A new cohort could, 15 Mørk SK, et al. J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access therefore, benefit from the observed improvement of the vaccine prediction tool. In conclusion, personalized immunotherapy is a prom- ising approach to cancer treatment. We demonstrated that EVX- 01- CAF09b, a personalized neopeptide vaccine at three different dose levels is safe and capable of elic- iting T- cell responses in a clinical setting where patients received concurrent standard- of- care immunotherapy. We detected both CD8+ and CD4+ T cell responses toward corresponding vaccine peptides and found that the number of immunogenic peptides and PIONEER epitope quality score are both predictive for good CR and longer PFS. Objective responses were observed in meta- static melanoma patients at all three dose levels, with all patients at the highest dose level achieving a CR. In addi- tion to the beneficial safety profile, EVX- 01- CAF09b may be a promising addition to ICI monotherapy. However, larger trials are warranted to confirm these findings and conclude on clinical benefit. Author affiliations 1Department of Oncology, Copenhagen University Hospital, National Center for Cancer Immune Therapy (CCIT- DK), Herlev, Denmark 2Department of Health Technology, Technical University of Denmark, Kgs. Lyngby, Denmark 3Medical Oncology, Radboudumc, Nijmegen, The Netherlands 4Evaxion Biotech, Copenhagen, Denmark 5Center for Vaccine Research, Statens Serum Institut, Copenhagen, Denmark 6Genomic Medicine, Rigshospitalet, Copenhagen, Denmark 7Department of Urology, Copenhagen University Hospital, Herlev, Denmark 8Department of Gastroenterology, Copenhagen University Hospital, Herlev, Denmark X Arianna Draghi @AriannaDraghi and Marco Donia @doniamarco Acknowledgements We thank all the patients who participated in the study. We are very grateful to the Kennedy Center (Genomic Center) staff for their support and assistance. In addition, we thank the staff at Gastroenheden Herlev for performing needle biopsies; the staff at the Department of Urology at Herlev Hospital for performing ultrasound- guided intraperitoneal vaccinations; the staff at Evaxion for designing the personalized neoantigen vaccines (PIONEER operations team), GMP manufacturing of EVX- 01- ds (CMC team) and assistants in running a few ELISpot assays; the laboratory technicians at CCIT- DK for invaluable help with handling blood samples and the vaccine preparation; the staff at SSI for data analysis and data interpretation; the Department of Oncology, Herlev Hospital, physicians and nurses for their support and loving care of the patients. We thank PhD Tripti Tamhane and Anna Dinna Ester Gyllenberg Burkal for production of MHC class I monomers.