J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access mm3 fragments and placed in separate wells of a 24 well- culture plate with medium (90% RPMI- 1640 plus GlutaMAX and 25 mM HEPES), 10% HS, 100 U/mL penicillin, and 100 µg/mL streptomycin, 1.25 µg/mL Amphotericin B and 6000 IU/mL rhIL- 2, for 3–6 weeks. Minimally expanded TILs were then pooled and either cryopreserved or further expanded according to a standard 14- day REP.21 Autologous tumor cell lines (TCLs) were established via serial passage of adherent cells from the same tumor biopsies.20 The TCLs were routinely tested negative for mycoplasma (AppliChem; Darmstadt, Germany), and the number of passages between collection and use in the described experiments was <10. T cell activation assay by IFN-γ ELISPOT Using the IFN-γ ELISPOT assay, we screened for peptide recognition by T cells in both PBMCs and SKILs as described in the interim report.9 T cell activation assay by intracellular cytokine staining analyses PBMCs (peptide-specific activation) Rested PBMCs were cultured in a 96- well plate (2–3×106 cells/well) and restimulated with the vaccine peptide pool (20 µg/mL) or irrelevant peptide (1 µg/mL) in X- vivo media and incubated at 37°C. After 2 hours, Golgi mix containing GolgiPlug, GolgiStop (BD biosciences), and CD107a (BD biosciences) were added and incubated for 6 hours. The cells were washed and stained with Live/Dead Fixable Dead Cell Stain Near- IR (Thermo Fisher) and Surface antibodies: CD3, CD4, and CD8 (BD biosciences) and fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (Invit- rogen). Next, the cells were intracellularly stained for TNF-α (Biolegend), IFN-γ , and CD137 (BD biosciences). Cells were analyzed using the LSRFortessa (BD biosciences), and flow cytometry data were analyzed by FlowJo V.10 (Becton Dick- inson). Reactivity of the T cells was defined as a percentage of live CD8+or CD4+ T cells staining positive for at least two of four reactivity markers (TNF-α , IFN-γ , CD137, and CD107a). Irrelevant peptides with effector cells were used as control. Patients were defined as having a vaccine- specific response when the frequencies of reactive T cells stimulated with EVX- 01 neopeptide pool exceeded the frequency of reactive T cells stimulated with irrelevant peptide. SKILs and TILS (peptide-specific activation) Prior to the initiation of the assays, SKILs and TILs were thawed and rested overnight. EVX- 01- specific T cell activation was evaluated with an 8- hour co- culture at 37°C of effector cells (TILs or SKILs) and peptides in the presence of autologous monocytes. The SKIL to monocyte ratio was 10:1. The single peptides and peptide pool were added with a final concen- tration of 0.5 µg/mL alongside a positive control (PMA+lon- omycin) and a negative control (irrelevant peptide). After 2 hours of co- culture, anti- human CD107a antibody, brefeldin A (dilution of 1:1000, GolgiPlug), and monensin (dilution of 1:1000, GolgiStop) were added. After 8 hours of incubation, the cells were washed two times with DPBS (Sigma- Aldrich/ Merck KGaA) and stained with live/dead reagents, as well as antibodies used for surface markers. Afterward, the cells were washed, fixed and permeabilized overnight using the FoxP3/Transcription Factor Staining Buffer Set (eBiosci- ences, Thermo Fisher Scientific). Following, the cells were stained with antibodies binding to intracellular targets. Cells were then analyzed on a NovoCyte Quanteon Flow Cytom- eter with FlowJo V.10 (Becton Dickinson). The T cell reac- tivity was defined as the percentage of live CD8+ or CD4+ T cells staining positive for at least two of four reactivity markers (TNF-α , IFN-γ , CD107a, CD137) minus the background (unstimulated control). Further details can be found in the interim manuscript presenting interim data.9 SKILs (tumor-specific activation) Prior to the initiation of the assays, SKILs were thawed and rested overnight. SKILs were then tested for antitumor reactivity as previously described.22 The definition of T cell reactivity was the percentage of live CD8+ or CD4+ T cells staining positive for at least two of four markers (TNF-α, IFNγ, CD107a, CD137) minus the background (unstimulated control). Detection of peptide-specific CD8+ T cells by combinatorial fluorochrome encoding of peptide-MHC multimers 8–11 mer minimal peptides were predicted from every EVX- 01 peptide (22–27 mer) by NetMHCpan V.4.1,23 based on patient- specific HLA types. EVX- 01- derived minimal peptides were selected per patient based on EL%rank <2. A selection process was performed to reduce the number of peptides; we selected the top 30–41 peptides with lowest EL%rank, grouped by the long EVX- 01 peptides and the patient- specific HLA alleles (online supplemental figure 6). A few EVX- 01 peptides did not include HLA class I binders (EL%rank <2), which is why no minimal peptides were included for these peptides. A panel of fluorochrome- labeled peptide- MHC (pMHC) tetramers was assembled for each patient, with each pMHC having a unique identifiable fluorochrome combination, as previously described.24 25 EVX- 01 prestimulated PBMCs for all collected time points were stained with the patient- specific pMHC tetramer panel, analyzed on an LSR fortessa (BD Bioences) and gated in FlowJo V.10. Detection of peptide-specific CD8+ T cells using DNA barcode-labeled pMHC I multimers Selected patients (patients 1, 2, 3, 4, 8, and 9—primarily patient with a CD8 response detected by intracellular cyto- kine staining (ICS)) were screened for the broad presence of neoantigen reactive CD8+T cells (NARTs) and virus- reactive CD8+T cells (VARTs). Patient- specific neopeptides were predicted with two different prediction pipelines, PIONEER and MuPeXi.26 To construct patient- specific peptide libraries, the top 100 peptides from each prediction pipeline were selected (including potential overlapping peptides, resulting in <200 peptides per library). Additionally, EVX- 01 vaccine minimal peptides described above and virus peptides were added to the patient- specific panel of peptides resulting in a total of 145–231 unique pMHC combinations per patient. 7 Mørk SK, et al.