The total number of nAgs for the analyzed tumor biopsies is shown inFig. 1C . For 1 patient (Pt 6), the tumor biopsy displayed a total number of 34 neoantigens. Therefore, in this case, the 34 neoantigens were all encoded in the NOUS-PEV vaccine and not subject to the VENUS selection (11), as the cumulative length of the sequence corresponding to the candidate neoantigens was lower than the maximum capability of the gene expression cassette. For the remaining patients, the best subset of neoantigens were all selected by VENUS, which ranked the total detected nAgs and selected the top ranked mutations up to the maximum capability allowed by our viral vectors (/C2460 25-mer neoantigens; ref. 8). An overall number of 398 of 1591 identi ﬁed nAgs were selected by the prioritization algorithm. The selected nAgs were retrospectively classi ﬁed according to arbitrary deﬁned thresholds for the three parameters used by VENUS for neoAgs selection: (i) the likelihood of presentation by MHC-I; (ii) the expression and (iii) the allele frequency of the somatic mutations encoding nAgs as detailed in Fig. 1D . The nAgs present in the vaccines were enriched for having high scores in these three para- meters, with 92% of the selected nAgs (365 of 398) displaying at least one “good ”parameter; 52% had at least two out of three “good ” parameters ( Fig. 1D ).NOUS-PEV is safe and induces T-cell responses against neoantigens in all evaluable patients The vaccination was safe and well tolerated. No treatment-related serious adverse events (SAE) or Grade 3 and 4 (CTCAE) AEs occurred. AEs related to NOUS-PEV (attributed by investigator to NOUS -PEV alone or NOUS-PEV and pembrolizumab) were reported in four (66.7%) patients, all of which were Grade 1 or 2. The most common AEs related to NOUS-PEV were localized injection site reactions (50%, n¼3;Table 2 ). Of the 6 patients who received vaccination and pembrolizumab, the best overall responses (BOR) observed were one complete response (CR), three partial responses (PR), one stable disease (SD), and one disease progression (PD; Table 1 ). Vaccine immunogenicity was an exploratory endpoint of the study and was evaluated by an IFN gex vivo enzyme-linked immunosorbent spot (ELISpot) assay using 6 patient-speci ﬁc peptide pools ( /C2410 peptides per pool) covering the entire vaccine sequence ( Fig. 2A ). For the assessment of immune response, peripheral blood mononu- clear cells (PBMC) were collected at different time points before and after vaccination. Stimulation of PBMCs with patient-speci ﬁc peptide pools resulted in IFN gT-cell responses post-vaccination in 100% of evaluable patients who had received the prime/boost regimen ( n¼4; Fig. 2B ). Interestingly, immune response was not detected in Pt 5, who did not receive GAd20 but only MVA. None of the patients had a positive ex vivo IFNgELISpot response to their cognate neoantigen peptide pools after pembrolizumab, prevaccination administration (Fig. 2B ). Total response at peak after vaccination reached a mean of/C24700 spot-forming cells (SFC)/million PBMCs. Responses were directed against different peptide pools, with the induction of a polytope response to the vaccine neoantigens ( Fig. 2C ). Antigen- speciﬁc expansion of T cells after in vitro stimulation (IVS) resulted in additional positive responses by PBMC samples collected after vac- cination against pools with undetectable ex vivo response (Supple- mentary Fig. S1). Depletion of CD8 cells from PBMC showed that vaccine induced T-cell responses included both CD4 and CD8 T cells (Fig. 2D ). Assessment of individual CD4 and CD8 T-cell speci ﬁcity against each peptide was not feasible for all patients and for all the peptide pools, given the considerable number of encoded neoantigens coupled to the limitation in sample availability. Vaccine-induced T- cell responses were long lasting, as we found that cellular immunity was still detected 7 months after vaccination ( Fig. 2E ). TCR neoantigen-speci ﬁc T-cell repertoire is expanded and diversi ﬁed after NOUS-PEV vaccination Clonal dynamics of antitumor TCR clonotypes before and after treatment were next investigated. More speci ﬁcally, the intratumoral TCR repertoire from bulk RNA-seq in matched pre- and posttreatmentTable 1. Demographic and disease features of the NOUS-PEV study population. Patient ID 1 2 3 4 5 6 7 Age 64 72 77 88 74 62 72 Sex Male Male Male Female Female Male Male ECOG Grade 0 Grade 0 Grade 1 Grade 1 Grade 1 Grade 1 Grade 0 BRAF mutation Negative Positive Positive Negative Not applicable Negative Disease status Stage III Stage IV Stage III Stage III Stage III Stage III Stage IV BOR PR PR PD SD PR CR PD Sum of RECIST measurements at baseline 12 mm 100 mm 26 mm 10 mm 31 mm 46 mm 81 mm No. of target lesions 1 3 1 1 1 2 4 No. of nontarget lesions 1 2 1 1 1 3 2 Abbreviation: ECOG, Eastern Cooperative Oncology Group.NOUS-PEV Vaccine Elicits Potent Antitumor T-Cell Responses AACRJournals.org Clin Cancer Res; 30(11) June 1, 2024 2415Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 Figure 1. NOUS-PEV neoantigen vaccines: trial design, feasibility, and selection of encoded neoantigens. A,Schematic outlining the NOUS-PEV trial design, vaccine production, and treatment schedule. B,Achieved (triangles) and expected (green dotted line) turnaround time (TAT) to manufacture and release GAd20 and MVA- PEV. The TAT is calculated in days, from when the biopsy is available for the nucleic acid extraction till the day the vaccine is released. C,Total number of neopeptides detected in NOUS-PEV patients. Each bar represents the total number of neopeptides encoded by nonsynonymous somatic mutations detected in baseline tumor biopsies. Black and gray bars indicate the patients ’mutation subjected or not to VENUS prioritization algorithm, respectively. D,Quality of neopeptides included in NOUS-PEV. The pie chart displays the total number of candidate neopeptides targeted by NOUS-PEV. In green, blue, and salmon are indicated respective ly, the selected neopeptides showing one, two, or three of the parameters de ﬁned “good ”according to the following thresholds (TPM of the RNA carrying the mutation ≥1; MHC class I predicted binding IC 50≤500 nmol/L; mutation allele frequency >50%). In gray are the other neopeptides not included in the previous three categories. The bar plot indicates the detail of candidate neopeptides for each individual patient.D’Alise et al. Clin Cancer Res; 30(11) June 1, 2024 CLINICAL CANCER RESEARCH 2416Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 tumors was analyzed in 4 patients whose pre- and posttreatment biopsies were available (Pt 1, Pt 2, Pt 4, and Pt 5). Three out of 4 of these patients (Pt 1, Pt 2, and Pt 4) received the vaccine prime and boost regimen, whereas Pt 5 only received MVA and did not mount a detectable positive immune response in periphery post- vaccination ( Fig. 2B ). Analyses of intratumoral lymphocyte popu- l a t i o n ss h o w e da ni n c r e a s eo fTc e l l sm e a s u r e da sn u m b e ro fT C R b copies of nearly three-fold on average in posttreatment biopsies versus baseline in 3 patients who received prime and boost NOUS- PEV vaccine ( Fig. 3A andB). Interestingly, the intratumoral TCR repertoire of Pt 5, in the absence of full regimen vaccination, did not show expansion of T-cell repertoire as observed for the other 3 patients, suggesting that full r egimen heterologous prime boost vaccination is required to achieve T-cell expansion, in keeping with the lack of T-cell response measu red in the periphery. Consistent with the increase of the number of T-cell clones after NOUS-PEV, a more diverse TCR repertoire was also found in the three prime andboost vaccinated patients, suggesting ampli ﬁcation and diversi ﬁ- cation of antitumor T-cell responses posttreatment ( Fig. 3C ). To investigate the migration of individual neoantigen-speci ﬁc vaccine-induced CD8 T cells from the peripheral blood to the tumor, we focused on Pt 1, who showed a strong CD8 T-cell response detected ex vivo with PBMC samples post-vaccination. In this patient, elicitation and increase of vaccine-induced neoantigen T cells over time coincided with the deepening of the clinical response ( Fig. 4A ). Deconvolution of pool 1 in this patient revealed that the immune response was directed against one strong CD8 neopeptide, mutated NOL9 (named NeoAg7). NeoAg7 mutation was found in about 70% of tumor cells according to the mutation clonality analysis in the baseline biopsy, with a good predicted binding of the nonamer sequence RDLSIFSYL to the patient haplotypes HLA-B/C337:01 (Fig. 4B ).In vitro expansion of PBMCs collected after pembrolizu- mab (week 10) and vaccine treatment (week 14), in the presence of the NeoAg7 peptide, led to the expansion of NeoAg7-speci ﬁcC D 8 T cells as shown by the higher number of IFN gþSFCs ( Fig. 4C ). Unstimulated (DMSO only) cells from the same cultures served as negative control. Expanded NeoAg7-speci ﬁc CD8 T cells were then subjected to TCR bsequencing and compared with unexpanded T cells from the same cell culture conditions (week 14 PBMC). Neoan- tigen 7-speci ﬁc TCR clonotypes were then tracked in the patient tumor biopsies (baseline and on-treatment). Six NeoAg7 reactive TCRbclones expanded after IVS were detected in the tumor biopsies by bulk RNA analysis, with ﬁve clones exclusively detected in the posttreatment biopsy ( Fig. 4D ). Together, these results indicate that neoantigen-speci ﬁc CD8 T cells induced by NOUS-PEV vaccine expand and diversify upon treatment. Moreover, the presence of clonotypes from neoantigen-speci ﬁc CD8 T cells in tumor tissue that were also detected in PBMCs provides proof- of-concept that vaccine-induced neoantigen-speci ﬁc T cells in the periphery are able to traf ﬁc and in ﬁltrate into the tumor. Evaluation of biomarkers predictive for clinical response to NOUS-PEV and anti-PD-1 combination treatment Transcriptomic analysis of baseli ne biopsies from patients treat- ed with CPI allows the identi ﬁcation of potential mechanisms underlying tumor response and resistance (21). Recently, Cui and colleagues have shown that the ratio of IFN ggene signature to an immunosuppression signature (IMS) is a good predictor of the clinical response to CPI immunotherapy in patients with metastatic melanoma (18). This ratio was evaluated on a public dataset of tumor RNA expression data in patients with melanoma treated with CPI in parallel with our NOUS-PEV patient cohort. In this analysis, patients of the benchmar k cohort were divided into three categories: responding patients (R; including CR and PR); patients with stable disease (SD); and patients in progression (PD). We noticed that none of the patients with a clinical response in the public cohort had an IFN g/IMS ratio lower than /C01, different from t h eP Do rS Dp a t i e n t s( Fig. 5A ). NOUS-PEV patients were thus arbitrarily separated into two subgroups, one with a ratio IFN- g/IMS below /C01 (Pt 1, 3, 4, and 7) and the second group with a ratio higher than /C01 (Pt 2, 5, 6; Fig. 5A ). According to this analysis, patients 2, 5, and 6 fall under the group of predicted good responders to the pembrolizumab monotherapy, whereas patients 1, 3, 4, and 7 were predicted as subjects very unlikely to respond to pembrolizumab. Indeed, Pt 3 and Pt 7 rapidly progressed with Pt 7 progressing even before receiving the vaccine. Instead, Pt 1 showed an increase of tumor volume b efore vaccination at the ﬁrst CT scan but then a deep and durable response after vaccination. We thenTable 2. Treatment-Related AEs (TRAE) in patients treated with NOUS-PEV and pembrolizumab. Grade 1/2 Preferred term n(%) NOUS-PEV treatment-related AEsa Injection site reaction 3 (50) Fatigue 1 (17) Headache 1 (17) Hyperthyroidism 1 (17) Inﬂuenza-like illness 1 (17) Injection site erythema 1 (17) Injection site hypersensitivity 1 (17) Musculoskeletal stiffness 1 (17) Pruritus 1 (17) Pembrolizumab treatment-related AEsb Pruritus 4 (67) Diarrhea 3 (50) Fatigue 3 (50) Dyspnea 2 (33) Hyperthyroidism 2 (33) Hypothyroidism 2 (33) Injection site reaction 2 (33) Lipase increased 2 (33) Blood iron decreased 1 (17) Cough 1 (17) Dermatitis acneiform 1 (17) Dry mouth 1 (17) Dysgeusia 1 (17) Memory impairment 1 (17) Myalgia 1 (17) Nausea 1 (17) Papule 1 (17) Photopsia 1 (17) Rhabdomyolysis 1 (17) Vitiligo 1 (17) Note: Summary of number ( n) and percentage (%) of treatment-related AEs (TRAE). AEs were coded using MedDRA version 23.1. No patients had TRAE with CTCAE grade 3 or higher. Two patients had CTCAE grade 3/4 SAEs (1 patient had vertigo and 1 patient had hydronephrosis) all considered to be unrelated to the treatment. aNOUS-PEV –related AEs include events attributed by the investigator to NOUS- PEV alone as well as events attributed to both NOUS-PEV and pembrolizumab. bAEs related to pembrolizumab include events attributed by the investigator to pembrolizumab alone and events attributed to both NOUS-PEV and pembrolizumab.NOUS-PEV Vaccine Elicits Potent Antitumor T-Cell Responses AACRJournals.org Clin Cancer Res; 30(11) June 1, 2024 2417Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 Figure 2. NOUS-PEV elicits a potent neoantigen T-cell response. A,Schematic overview of ex vivo IFNgELISpot assay to measure immune response on PBMCs stimulated with 6 patient-speci ﬁcp e p t i d ep o o l sP 1t oP 6( /C2410 peptides per pool) covering the entire vaccine sequence. B,T-cell response measured in NOUS- PEV –vaccinated patients by ex vivo IFNgELISpot on PBMCs collected pre- and p ost-vaccination. Numbers of IFN gspot-forming cells (SFC) per 106PBMCs are shown for 4 patients receiving the full regimen GAd and MVA prime/boost (solid dots), and 1 receiving MVA-PEV (empty dot), comparing the baseline (pos t- pembrolizumab) responses versus the post-va ccination response at peak. Two-tailed Mann –Whitney test was performed (/C3,P<0.05). C,T-cell response measured in NOUS-PEV evaluable patients receiving the full regimen GAd/MVA (Pt 1, 2, 4, 6). Shown are the ex vivo immune responses measured post- pembrolizumab versus the post-vaccine immune responses against the peptide pools (P1 –P6) covering the entire vaccine sequence for 4 patients (Pt 1, 2, 4, 6). Graphs show mean SFC /C6SEM per 106PBMCs for triplicate ELISpot wells. Pools showing positive after ex vivo and in vitro restimulation (IVS) cultures are indicated with a “þ”symbol. Pie charts on the top represent the frequency of peptide p ools inducing CD8 and CD4 responses on the total pools eliciting a positive response by ex vivo /IVS ELISpot for each pati ent (NA, not available). D,Ex vivo IFNgELISpot on PBMCs before or after depletion of CD8þT cells in the presence of patient-speci ﬁc neopeptide pools. “CD4 ”and “CD8 ”indicate the subtype-speci ﬁc CD4 and CD8 T-cell responses. An unpaired ttest was used to detect signi ﬁcant differences between the grou ps (pre- and post-CD8 depletion);/C3/C3/C3/C3,P<0.0001;/C3/C3,P<0.01;/C3,P<0.05. E,Representative wells from IFN g ELISpot assay of PBMCs analyzed 7 months post-vaccination. The dimethylsu lfoxide (DMSO) wells represent the negative control, whereas a pool of vir al peptides (CEFX) was used as positive control.D’Alise et al. Clin Cancer Res; 30(11) June 1, 2024 CLINICAL CANCER RESEARCH 2418Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 investigated additional signatures aiming to identify factors that could characterize and diversify Pt 1 in clinical response from the others with PD. Loss/downregulation of MHC-I e xpression, as well as other key components of the MHC-I antige n processing machinery (APM), is a frequent immune evasion mechanism exploited by many cancers to escape CTL-mediated tumor cell killing. As low APM also hampers the outcome of CPI and other immunotherapiesincluding vaccines, we investigated the expression levels of key APM genes in NOUS-PEV responders (R; Pt 1, Pt 2, Pt 6) and PD p a t i e n t s( P t3a n dP t7 ) .T h eS Dp a t i e n t ,b e i n go n l yo n ea tt h e moment, was excluded from this analysis. Volcano plot depiction of differentially expressed g enes between NOUS-PEV PD versus R patients highlighted a signi ﬁcant downregulation in the set of APM genes, such as NLRC5, TAP1, HLA PSMB8 genes, in the PD group ( Fig. 5B ).