J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access vaccine starting clinical testing at CCIT- DK19 Only if no vaccine- related grade 3–4 AEs occurred in relation to the first two vaccinations at each dose level, the next patient could receive the first vaccination. The study’s primary endpoints were safety and tolera- bility of the treatment at each dose level, based on the observation of AEs according to the NCI Common Termi- nology Criteria for Adverse Events (CTCAE V.4.0). The secondary endpoints were the feasibility of manufacturing a personalized neoantigen- based vaccine within 6–8 weeks of inclusion via the PIONEER AI prediction platform and evaluating the immune response before, during and after vaccination with the personalized neoantigen vaccine (EVX- 01- CAF09b). The tertiary endpoints included effi- cacy, which was evaluated by best overall response (BOR), progression- free survival (PFS), and overall survival (OS). Assessments in the study included physical examina- tion, ECOG performance status, vital signs (pretreatment and post- treatment), and laboratory analyses to warrant the safety of the participants. Imaging (CT scan or PET- CT) was done at baseline, and every 3 months, followed by imaging every 12 weeks to evaluate the clinical efficacy of the trial treatment. Tumors were evaluated following the RECIST V.1.1 criteria. Tumor biopsies were obtained at baseline (obligatory), shortly before the initial vaccina- tion, and just after the final vaccination (voluntary). Design of personalized neoantigen vaccines Personalized neoantigen vaccines were designed by the PIONEER platform. WES data from healthy and tumor tissue, mRNA sequencing data from tumor tissue, and human leucocyte antigen (HLA) typing data from healthy tissue were used to predict the neoantigens and design the neopeptides. The PIONEER system assigns a combined neoantigen quality score to each neoantigen/neopeptide by combining (1) the potential to be presented by MHC, (2) expression levels, and (3) the clonality. The manufac- turing of the personalized neoantigen vaccine is described in detail in Mørk et al.9 In some patients, it was decided to administer individual neopeptides in double dose, that is, some individual neoantigens in dose level 1 were given at 100 µg/dose instead of target 50 µg/dose, 200 µg/dose instead of target 100 µg/dose at dose level 2 and 400 µg/ dose instead of target 200 µg/dose at dose level 3. This was accomplished by administrating a lower number of neopep- tides while keeping the total peptide dose level constant between patients at the same dose level. Patient 1, patient 3, patient 5, patient 6 and patient 10 had 4, 1, 1, 4 and 2, peptides administrated, respectively, in double dose as the target of manufacturing 10 soluble peptides could not be met. For patient 7, patient 8 and patient 9, it was decided to administer 5, 5, 4; peptides in double dose, respectively, to test higher individual peptide doses earlier in the trial. Each EVX- 01 vaccine comprises 5–10 synthetically manufactured peptides. The initial individual peptide dose was chosen based on the experience from a clinical trial performed at DK- CCIT (EudraCT No.: 2015- 003719- 39).19CAF 09b and final vaccine formulation at all dose levels EVX- 01 vaccine peptides were solubilized in 100% dimethyl sulfoxide (DMSO) (NPV- dp001) ad- mixed with the CAF09b adjuvant. CAF09b is a cationic liposomal vaccine adjuvant produced by Statens Serum Institut and is based on dimethyl- dioctadecyl ammonium (DDA) combined with monomy- coloyl glycerol (MMG) and poly I:C. Formulation of each EVX- 01 vaccine with CAF09b was performed at CCIT within 2 hours before administration as follows; a total of 1.08 mL sterile filtered Tris reconstitution buffer was added to 0.12 mL sterile filtered NPV- dp001 and thoroughly mixed. Following this step, 1 mL of the peptide solution was added to a 2R vial containing 1.0 mL CAF09b (2500 µg DDA/mL, 500 µg MMG/mL, and 125 µg poly I:C/mL). Subsequently, the final vaccine product could be drawn into a syringe. At dose level 1, patients received 500 µg of total peptide and 0.25 mL CAF09b, administered as 1×0.5 mL (IM) or 0.5 mL (IP). At dose level 2, patients received 1000 µg of total peptide and 0.5 mL CAF09b, administered as 2×0.5 mL (IM) or 1.0 mL (IP). At dose level 3, patients received 2000 µg of total peptide and 1.0 mL CAF09b, administered as 2×1.0 mL (IM) or 2.0 mL (IP). Peripheral blood mononuclear cells isolation and prestimulation Peripheral blood mononuclear cells (PBMCs) were obtained from peripheral blood at different time points (see figure 1A). Before screening for peptide recognition, PBMCs were prestimulated with pools of patient- specific EVX- 01 peptides. PBMCs were thawed and cultivated in X- vivo media (X- vivo 15, Lonza # + 5% human AB serum) supplemented with 10 ng/mL IL- 15 and 50 ng/mL IL- 21 (Preprotech) as culture media. At day 1, pooled vaccine peptides (final concentration 20 µg/mL) were added to the cell cultures in base media. From day 2, the culture media was supplemented with 40 IU/mL IL- 2 (Prepro- tech). The cells were cultivated for 10–14 days and rested for 1–3 days in X- vivo media before cryopreservation and subsequent analysis. Skin-test infiltrating lymphocytes A voluntary delayed- type hypersensitivity (DTH) skin test was done approximately 2 weeks after the sixth vaccinations (figure 2A). Patients received two intra- dermal injections of the EVX- 01 peptides and one injection with trisaminomethan buffer and DMSO as a negative control on the back. After 48 hours, 5 mm punch biopsies were obtained from each injection site for skin- test infiltrating lymphocytes (SKILs) culture. The tissue biopsies were divided into 1–3 mm3 frag- ments and cultured with medium (90% RPMI- 1640 plus GlutaMAX and 25 mM HEPES), 10% heat- inactivated AB Human serum (HS; Sigma- Aldrich), 100 U/mL penicillin, 100 µg/mL streptomycin (Pen Strep, Thermo Fisher Scientific), 1.25 µg/mL Amphotericin B (Fungizone, Bristol- Myers Squibb) and 100 IU/mL rhIL- 2 (Proleukin, Novartis). Plates 5 Mørk SK, et al.