J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access were incubated at 37°C, and 1/2 of the medium was replaced on day 5 and, hereafter, three times weekly. After 3–6, pooled SKILs were cryopreserved or further expanded with Rapid Expansion Protocol (REP).20Tumor-infiltrating lymphocytes and tumor cell lines As previously described,21 fresh tumor tissue was collected and transported to the laboratory for tumor- infiltrating lymphocytes (TILs) culture. In short, to obtain minimally expanded TILs, the tumor biopsies were divided into 1–3 Figure 2 EVX- 01 reactive SKIL derived T cell. (A) DTH skin test was done approximately 2 weeks after the last vaccination. Two intradermal injections of the EVX- 01 peptides and one control (DSMO) injection were administered on day 0 (approximately 2 weeks from the last vaccination). After 48 hours, 5 mm punch biopsies were taken from the three injections site. The tissue was then transported to the laboratory for SKILs culture. Minimally expanded SKILs were expanded from tissue fragments for 3–6 weeks. Created with BioRender.com. EVX- 01- specific T cell responses in SKILs (skin- test infiltrating lymphocytes) in patients 2 (dose level 1), 6, 7, 8 (dose level 2), and 9, 11 (dose level 3). (B) Elispot responses were examined in SKILs at T4 (after six vaccinations) from six patients. SKILs were co- cultured with EVX- 01 peptide pool and individual EVX- 01 peptides. Black bars represent significant responses, gray bars are not significant. The dotted line indicated the threshold value for a significant response; background (irrelevant peptide) plus 3×SD of the background and at least 10 spots over background response (C) T cells specific toward EVX- 01 peptides were identified in SKILs, which were restimulated with EVX- 01 peptide pool (green), single- vaccine peptides (green) or irrelevant peptide (pink) for 8 hours. The T cell reactivity was defined as the percentage of live CD8+ or CD4+ T cells staining positive for at least two of four markers (TNF-α, IFNγ, CD107a, CD137). 6 Mørk SK, et al.