J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access Figure 3 EVX- 01- specific PBMC derived T cell responses analyzed by Elispot and ICS. (A) Experimental setup for detection of T cells responses. PBMCs were prestimulated with EVX- 01 neopeptide pool for 10–14 days. Prestimulated PBMCs were hereafter stimulated with EVX- 01 neopeptides in an ELISpot assay or intracellular cytokine staining assay. (B) An overview of patient IFN-γ responses detected by Elispot grouped by dose levels and time points. Red dots represent dose level 1, green dots dose level 2, and blue dots dose level 3. The height of the dots on the y- axis represents the fraction of single vaccine peptides with a T cell response out of total neopeptides per patient. A total number of single peptides (N) are stated under the patient number at the X- axis. The size of the dots represents the sum of the spot after subtraction of the background (Irrelevant peptide). This only includes spots from single peptide stimulation, which induces positive responses. Gray scattered boxes indicate time points which were not analyzed. (C) Representative ELIspot wells for patient 10 pre vaccination (T1 and T2), and post vaccination (T3 and T4). (D) The individual vaccine dose of the single EVX- 01 peptides versus delta spots for peptides with T cell response (background stimulated with irrelevant peptide has been subtracted). Responses have been divided in pre vaccination (T1 and T2), postvaccination (T3 and T4) and follow- up (all FU time points). Each dot represents a single- peptide response detected with Elispot. Means were compared between peptide dose groups using Kruskal- Wallis test. Significant difference is indicated with asterisks (*p<0.05, **p<0.01). (E) T cell responses was tested after restimulation with pool vaccine peptides and stained intracellularly for IFN-γ, TNF-α, CD107a and CD137. The percentages of T cells which are positive for at least two of mentioned markers are shown in an overview of EVX- 01- specific CD4+ and CD8+ T cells in all patient responses from all three dose levels and all time points. Red bars represent dose level 1, green bars dose level 2, and blue bars dose level 3. Bars show specific CD4+ and CD8+ T cells. Background response has been subtracted. Gray scattered boxes indicate time points which were not analyzed. (F) Flow cytometry dotplots for CD4+ and CD8+ T cells stained for IFN-γ, TNF-α, CD107a and CD137. The dotplots are shown for patient seven with the highest CD4+T cell response at T4, and patient eight with the highest CD8+T cell response at T4. ICS, intracellular cytokine staining; PBMC, peripheral blood mononuclear cell. 10 Mørk SK, et al. J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access The fraction and magnitude of neoantigen- induced ELISpot responses are depicted in figure 3B. Overall, fewer and of lower magnitude pre- existing responses were detected before initiation of EVX- 01 vaccination (T1 and T2). Post EVX- 01 vaccination (T3 and T4), neoantigen responses were observed in all patients with a significant increase in response magnitude (sum of spot forming units) compared with base- line samples. Furthermore, ELISpot responses were detected in all follow- up samples showing that vaccine- induced T cells persisted in patients for up to 14 months from the last vacci- nation (last FU- sample time point). However, a decrease in both frequency and magnitude was observed over time (figure 3B,C, online supplemental figure 2). To investigate the effect of EVX- 01 peptide dose, the magni- tude of T cell responses was plotted against the dose of the individual peptides (figure 3D, online supplemental figure 3). It could be observed that higher peptide dose correlates with T cell response magnitude and peptides administered in doses of 200 µg induced a significantly higher response (number spot forming units, SFU, background subtracted) than peptides delivered with a dose of 50 µg, maintained during follow- up. EVX-01 induces both CD4+ or CD8+ T-cell responses We evaluated whether the EVX- 01- specific T- cell responses were CD4+ or CD8+ T cell- mediated. Therefore, EVX- 01 prestimulated PBMCs were restimulated with patient- specific EVX- 01 peptide pools and intracellularly stained for IFN-γ , TNF-α , CD137, and CD107a to detect T- cell reac- tivity against the peptide pool using flow cytometry. EVX- 01- reactive CD4+T cells were observed in all 12 patients after vaccine administration at T3 and T4 (figure 3E,F). CD4+T cell responses persisted in follow- up samples, however, the frequency of reactive CD4+T cells decreased over time. CD8+T cell responses were observed in seven patients in total; five patients at T3 and T4 and additional two patients at FU1 (figure 3E,F). In general, minor reactivity was detected before vaccination (T1 and T2) for both CD4+ and CD8+ cells. There was no obvious difference in reactivity between the three dose levels. As opposed to ELISpot results, ICS results were only based on EVX- 01- peptide pool stimulation and not individual EVX- 01- peptide stimulation. EVX-01-induced T cells can migrate into solid tissue As antigen- specific homing of immune cells to the tumor tissue is important for tumor cell elimination, the homing capacity of EVX- 01 induced T cells was analyzed by a DTH skin test in six patients (patients 2, 6, 7, 8, 9 and 11, figure 2A). We evaluated the presence of EVX- 01- specific T cells in SKILs by IFN-γ ELISpot and ICS following peptide stimulation. We observed reactivity against indi- vidual EVX- 01 peptides in the SKILs from four patients by ELISpot after EVX- 01- CAF09b vaccination (figure 2B). By use of ICS, we could confirm the presence of CD4+T cell responses in these 4 patients and a CD8+T cell response in the SKILs of one of these patients. Furthermore, a low frequency of EVX- 01- specific response was detected in CD4+T cells in the SKILs of patient 9, not picked up with IFN-γ ELISpot (figure 2C, online supplemental figure 6). Responses to single peptides in ICS corresponded to responses detected in SKILs using IFN-γ ELIspot. The specific responses observed in SKILs had also been detected in PBMCs with Elispot, except for peptide 2 and 4 detected in SKILs from patient 6 (online supplemental figure 2). For patient 6, a TCL was established from a tumor biopsy collected before initiation of EVX- 01- CAF09b vaccination (T2). In this patient, SKILs were evaluated for tumor reactivity, but no tumor recognition was observed (data are not shown). In summary, EVX- 01 SKIL reactivity analyzed in Elispot and ICS assays is comparable to our results from EVX- 01 prestimulated PBMCs. Additionally, tumor biopsies were collected both before (T1 and T2) and after (T4) vaccination from three patients (patients 1, 6, and 7). TILs were success- fully expanded from all tumor biopsies, but no reactivity was detected when these TILs were exposed to EVX- 01 peptides(data are not shown). Of note, patients 6 and 7 did not respond to treatment and, in general, had low- quality neoepitopes (see paragraph below). Vaccine-related neoantigen recognizing CD8+ T cells were detected in prestimulated and ex vivo PBMCs EVX- 01 prestimulated PBMCs were screened for CD8+T cells that recognize minimal epitopes within the EVX- 01 vaccine peptides. CD8+T cells recognizing minimal peptides predicted from EVX- 01 were defined as VaccNARTs (vaccine- embedded neoantigen- recognizing CD8+T cells). The presence of VaccNARTs was exam- ined by staining with pMHC I tetramers. Any overall increase in VaccNARTs frequency after vaccination was not detected when summarizing the signal across patients (online supplemental table 1, figures 7 and 8). However, an increase in VaccNARTs was observed in selected patients after vaccination. In patient 6, a strong enhance- ment of low- level pre- existing and induction of de novo T- cell recognition was observed toward minimal peptides derived from vaccine peptide 2 and 6 (figure 4B). In patient 8, VaccNARTs were observed against a minimal peptide derived from vaccine peptide 3 (KLYASPSQFIK). This response was increased after ICI treatment T2 and again after IM vaccination T4 (figure 4B). Furthermore, using barcoded- pMHC multimers, we evaluated CD8+T cell recognition toward 145–231 HLA- binding neoepitopes predicted across the full tumor mutagenome, that is, also beyond those embedded in the Table 3 Elispot responses overview Dose levelTotal peptides (n)Total responsesDe novo responses All 91 53 (58%) 45 (49%|85%) Dose 1 41 26 (63%) 22 (54%|85%) Dose 2 16 11 (69%) 7 (44%|64%) Dose 3 34 16 (47%) 16 (47%|100%) % of total peptides % of total responses 11 Mørk SK, et al. J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access Figure 4 EVX- 01 prestimulated PBMCs and ex vivo PBMC derived CD8+T cells screened for EVX- 01 and other CD8+T cell neoepitopes. (A) A schematic overview of the experimental setup for detection of VaccNARTs in prestimulated PBMCs. Minimal peptides embedded in the EVX- 01 neopeptides were predicted and loaded on tetramers conjugated to fluorochromes. Specific combinations of two fluorochromes were used for identification of the specificities of detected VaccNARTs. (B) The frequency of selected VaccNART with only one specificity, from patients 6, 8 and 10. EVX- 01 peptide number (Vaccpep), short peptide sequence, HLA, and whether the short peptide includes the mutated region of the Vaccpep (mut) is shown above the graphs. dotplots for time point T1 (baseline) and T3 (after three IP vaccinations) are shown for each peptide. (C) A schematic overview of experimental setup used for detection of NARTs, VaccNARTs and VARTs in ex vivo PBMCs, expanded TILs and SKILs. CD8+T cells reactive toward predicted neoepitopes, including vaccine embedded neoepitopes, and virus epitopes were screened with DNA barcoded pMHC multimers. (D) The frequency VaccNARTs shown for selected patients, showing the single populations dynamics over time. EVX- 01 peptide number (Vaccpep), short peptide sequence and HLA is shown next to the graphs. IP , intraperitoneal; PBMC, peripheral blood mononuclear cell. 12 Mørk SK, et al. J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access EVX- 01 vaccine peptides. We analyzed CD8+T cells that recognize such predicted neopeptides and categorized as either VaccNARTs (recognizing vaccine- embedded sequences) or NARTs (recognizing non- vaccine- embedded neoepitopes) directly ex vivo on PBMCs and expanded TILs and SKILs from six patient. We did not observe any overall increase in frequencies of NARTs after vaccination, hence no signs of epitope spreading occurring after vaccination. However, an increase in the number of NARTs was observed after ICI for patient 1 (online supplemental figures 9 and 10). When evalu- ating the VaccNARTs using this methodology, we find that frequency and number generally appeared to increase on ICI administration (T2) and then again after EVX- 01 vaccination (T3) (figure 4C and online supplemental figures 9 and 10). New VaccNARTs detected in PBMCs after vaccination were also detected in TILs from tumor biopsies taken before vaccination, as seen for patient 1 and 8, and thus not a de novo response induced by the vaccine (figure 4C and online supplemental figure 9). This was supported by the observed recognition of an epitope for vaccine peptide 3 (KLYASPSQFIK) in patient eight prior to vaccinations (T2) in EVX- 01 prestimu- lated PBMCs, as mentioned above (figure 4B). Finally, VaccNARTs were detected in SKILs from patients 2 and 9, where patient 2 had a larger frequency of VaccNARTs in SKILs compared with TILs (T1 and T2), indicating a boost in tissue homing VaccNARTs by the vaccine. In general, VaccNART and NARTs were only detected in low frequencies, although the frequency and the number of virus- responsive T cells (VARTs) were stable over time, demonstrating assay stability between time points (online supplemental figures 9 and 10). CR correlates with neoantigen immunogenicity and PIONEER scores Next, we investigated how the PIONEER quality score of administered EVX- 01 neopeptides and the response to the vaccine correlated with clinical outcome. First, we compared the level of T cell responses with the BOR. We observed that patients who benefitted from treatment (PR or CR) responded to more EVX- 01 neopeptides compared with patients not benefitting from treatment (SD or PD) (figure 5A,B). The difference in immune response between responders and non- responders seems to increase over time, with no difference observed before vaccination and the highest difference observed at follow- up. However, among the responders, CR did not have an overall better vaccine response compared with PR (figure 5A,B). Furthermore, we observed significantly more de novo T cell responses detected by IFN ELIspot in patients with a CR (figure 5C). The observed correla- tion between immunogenicity appeared to be connected to the broadness of response as we did not find a correla- tion between clinical outcome and the magnitude of IFN-γ ELIspot response or the magnitude of ICS- detected responses (online supplemental figure 11).We then evaluated the impact of the PIONEER quality score assigned to each EVX- 01 administered neoantigen and used to select neoepitopes for treatment of each patient. As shown in figure 5D, the PIONEER quality scores are significantly associated with immunogenic EVX- 01 neopeptides. Although immune response toward cancer- specific epitopes is essential, this parameter alone does not dictate clinical efficacy. We, therefore, inves- tigated if the PIONEER quality score of administered EVX- 01 peptides could separate responders from non- responders, as depicted in figure 5E. In contrast to the magnitude of immune responses, the PIONEER quality scores are significantly higher for responders compared with non- responders. This might be attributed to the eval- uation of neoepitope abundancy, expression, clonality, and tumor evasion embedded in the PIONEER quality score additionally to evaluating the immune response of neoepitopes. Algorithms for identifying effective neoepitopes are a rapidly developing field.15 Since initiating the EVX- 01- CAF09b phase I clinical trial, we have progressed the prediction platform from version 2 to version 4. The development consists of improvements of submodules of PIONEER- 2 as well as the addition of new models. The PIONEER- 4 quality scores do more efficiently sepa- rate immunogenic from non- immunogenic peptides compared with the PIONEER- 2 quality scores with signifi- cantly higher scores assigned to immunogenic neoan- tigens (figure 5F). Additionally, VaccNART detected EVX- 01 peptides are all found within the highest predic- tion scores, except one. When comparing the PIONEER- 4 quality scores for neopeptides administered to patients, these are, as the for the PIONEER- 2 scores, highest for EVX- 01 peptides in CR (CR/PR) (figure 5G). Compared with PIONEER- 2 scores, the PIONEER- 4 scores are lower in patients with SD and PD. These interesting findings around the PIONEER quality score prompted us to investigate how the scores correlate with PFS. Interestingly, when separating the patient population based on high and low PIONEER- 4 median quality score (balanced, n=6 in each group), a significantly longer PFS in patients with high scores was observed (figure 5H). To investigate if the longer PFS in the high- score group was driven by a higher mutational load, the same analysis using TMB was conducted. As depicted in figure 5I, TMB did not seem to be the deter - mining factor for PFS in this patient cohort, indicating that the quality of administered EVX- 01 neopeptides is important for clinical benefit. DISCUSSION Here, we present the final data from the phase I clinical trial, “Personalized Neo- antigen Vaccine in Advanced Solid Tumors (NeoPepVac)”. In this study, patients with metastatic melanoma were treated with a person- alized neopeptide vaccine EVX- 01- CAF09b in addition to anti- PD- 1 therapy. The study addresses the safety, 13 Mørk SK, et al.