Figure 3. Increase of intratumoral T cells post- NOUS-PEV treatment with vaccine- induced T cells in ﬁltrating tumor. A,Expansion and diversi ﬁcation of TCRbrepertoire in pre- and posttreat- ment tumor biopsies in four NOUS- PEV patients (3 PR;1 SD). Each bar indicates the abundance of individual TCRbclones detected in the total tumor RNAs extracted from the biop- sies collected at the two time points (details in Patients and Methods). B,Total number of TCR bcounts detected in tumor biopsies estimated by summing up the abundance of the individual clones reported in A. The solid lines represent NOUS-PEV patients receiving the full vaccine reg- imen GAd/MVA; the dashed line represents the patient (Pt 5) who only received MVA-PEV. C,Number of indi- vidual clones with different TCR b CDR3 detected in the patients ’tumor biopsies (/C3,P≤0.05, paired ttest).NOUS-PEV Vaccine Elicits Potent Antitumor T-Cell Responses AACRJournals.org Clin Cancer Res; 30(11) June 1, 2024 2419Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 Discussion Tumor neoantigens represent the optimal target for the generation and ampli ﬁcation of immune responses by therapeutic vaccinationcombined with CPI treatment. In this manuscript, we report the feasibility and immunogenicity of NOUS-PEV assessed in a phase Ib study in patients with metastatic melanoma. NOUS-PEV is an indi- vidualized neoantigen vaccine based on heterologous GAd20/MVA Figure 4. Vaccine-induced neoantigen T cells migrate into the tumor. A,T-cell response assessed by ex vivo ELISpot versus percentage changes in target lesion size (sum of target lesion measurements evaluated per RECIST v1.1.) from baseline are displayed for NOUS-PEV Pt 1. B,Deconvolution of T-cell response by ex vivo ELISpot against individual peptides of the immunogenic pool 1. The immunogenic NeoAg7 is shown in black. The unpaired ttest was used to make comparisons (/C3/C3/C3/C3,P<0.0001). C,Top, schematic representation of the IVS protocol to expand vaccine induced neoantigen speci ﬁc T cells against NeoAg7 peptide. Bottom, T-cell responses in Pt 1 measured by IFN gELISpot assay after IVS with NeoAg7 peptide. Tested PBMCs were collected after pembrolizumab (week 10) and after vaccination (week 14). The unpaired ttest was used to make comparisons (/C3/C3/C3,P<0.001). D,Expansion and diversi ﬁcation of TCR brepertoire in pre- and posttreatment tumor biopsies of Pt 1. Each bar is a TCR bindividual clone; the clonotypes speci ﬁc for NeoAg7 detected on the tumor biopsies of Pt 1 are shown as black bars.D’Alise et al. Clin Cancer Res; 30(11) June 1, 2024 CLINICAL CANCER RESEARCH 2420Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 prime boost, in combination with pembrolizumab. We demonstrated that this personalized vaccine approach relying on two different viral vectors is feasible, given the fact that 90% of the NOUS-PEV products were successfully manufactured. Moreover, in all cases, vaccines were administered to the patients according to the prede ﬁned timeline with a median vaccine release of about 8 weeks from biopsy. Manufacturing speed is particularly relevant in a setting of metastatic disease but also in curatively resected disease to avoid unnecessary treatment delay and maximize the opportunity for clinical bene ﬁt. To date, depending on the choice of vaccine platform, the manufacturing time reported in the ﬁeld typically ranges from 8 to 16 weeks (22), positioning this approach among the more rapidly manufactured ones so far. Combination of NOUS-PEV with pembrolizumab was safe, with no treatment-related SAEs reported for any of the treated patients, and with only mild reactions to the vaccines, consistent with the results of other studies of heterologous prime/boost vaccine schedules incorporating GAd and MVA (7, 23). The vaccine approach presented here allows targeting an unprecedented number of neoantigens, with a strategy of polyvalent vectors encoding 60 patient-speci ﬁc neoantigens. Indeed, antigen lossand tumor heterogeneity represent important challenges to the effec- tiveness and durability of the antitumor response and several evidences indicate that the targeting of multiple neoantigens may enhance the rate of ef ﬁcacy (8). High-magnitude vaccine-induced immune responses detected byex vivo IFNgELISpot were elicited in all evaluable patients post-NOUS-PEV administration, with polytope responses measured against multiple peptides. These results are in line with those obtained in a different clinical trial using the same platform targeting frameshift (FSP) neoantigens shared across patients with dMMR and eliciting a potent and broad T-cell response in the vast majority of patients (9). Immune responses were both CD8 and CD4 T-cell responses, con- ﬁrming the capability of this platform to induce both types of T lymphocytes, as also observed in preclinical models (8, 11). One patient (Pt 5) who only received MVA instead of the full vaccine regimen, did not mount T-cell responses against vaccine encoded neoantigens, indicating that the combined GAd/MVA approach is required to mount proper T-cell immunity. Of particular interest from the clinical point of view is patient 1, who showed stable disease at the ﬁrst CT scan (after 3 pembrolizumab infusions) with dynamic target lesion growth, followed by tumor shrinkage only after vaccine admin- istration, resulting in a con ﬁrmed PR. Deepening of tumor shrinkage coincided with the increase of the vaccine induced immune response detected in the periphery. The potential lack of response to anti-PD-1 of this patient was in line with the predictive gene signatures evaluated from pre-treatment tumor biopsy. Indeed, this patient displayed a very low ratio of IFN gsignature to IMS, previously demonstrated to be indicative of a low probability of responding to anti-PD-1 monother- apy (18). Deconvolution of immune response at the level of single peptides was possible in this patient and allowed the identi ﬁcation of immunogenic neoepitope inducing the CD8 T-cell response. Inter- estingly, the identi ﬁed NeoAg (#7) is in the mutated nucleolar protein NOL9 and shares 100% sequence identity with a Lachnospiraceae peptide. Sequence homology between neoantigens and microbial peptides has been suggested to contribute to the induction of an immune response against neoantigens (24). Moreover, Lachnospir- aceae belongs to the group of Bacteroides previously shown to correlate positively with the rescue of clinical response to anti-PD-1 by fecal transplant (25). Enrichment of T cells in the tumor post MVA was detected via TCR sequence in all three evaluable patients receiving the full GAd20/MVA vaccine regimen. This ﬁnding is indicative of the NOUS-PEV vaccine ’s ability to expand and diversify T cells in the tumor, consistently with the TCR repertoire changes observed by the very same platform in MSI patients treated with Nous-209 off-the- shelf vaccine (9). Moreover, in patient 1, TCR clonotypes identi ﬁed as speciﬁc for NeoAg (#7) were tracked in the tumor biopsy and expanded and diversi ﬁed after NOUS-PEV vaccination. These results provided further evidence of tumor in ﬁltration by neoantigen-speci ﬁc T-cell clones following vaccination. Enrichment of T cells in the tumor correlates with the kinetics of T-cell expansion in the periphery post- vaccination. Previous ﬁndings have shown that expansion of T cells in the tumor due to CPI activity occurs very early post treatment initiation and therefore may no longer be detected at the biopsy collection time selected in this study, 13 weeks post-CPI treatment initiation. Interestingly enrichment of T cells in the tumor was not observed in Pt 5 in clinical response, who did not receive GAd20 priming and had no detectable immune response to NOUS-PEV in the periphery. To date, immunogenicity of neoantigen vaccines has been shown in patients with cancer to a varying extent using different technologies (26 –28). A recent study in patients with surgically Figure 5. Analysis of potential biomarkers predictive for antitumor response. A,Ratio of the IFN gsignature score to the IMS score predictive of PD-1 blockade (18) estimated according to RNASEQ gene expression values detected in pretreat- ment tumor biopsies. The values of the signature estimated in NOUS-PEV patients were compared with a dataset of 172 patients with melanoma (57 CR/PR; 28 SD; 87 PD) treated with anti-PD-1 monotherapy and retrieved from four published studies (18, 31 –33). B,Volcano plot of differentially expressed genes detected by analyzing the RNASEQ of pretreatment biopsies of patients in progression (PD; n¼2) versus the ones collected from responder patients ( n ¼3; median log2 FC </C01o r>1; Benjamini –Hochberg corrected Pvalue <0.05 according to a consensus of four different methods. Details in Patients and Methods). The plot highlights in red a subset of 21 antigen present machinery- related genes (APM) downregulated in NOUS-PEV PD patients.NOUS-PEV Vaccine Elicits Potent Antitumor T-Cell Responses AACRJournals.org Clin Cancer Res; 30(11) June 1, 2024 2421Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 resected pancreatic ductal adenocarcinoma (PDAC) tumors treated adjuvantly with mRNA vaccine encoding 20 neoantigens, in combi- nation with atezolizumab and mFOLFIRINOX, showed induction of T-cell responses in 50% of treated patients that correlates with delayed recurrence (28). Although a direct comparison between platforms is not feasible, as the clinical settings and indications targeted are different, it is interesting to note that immunogenicity of mRNA vaccine was detected in only half of the treated patients, whereas viral-based neoantigen vaccine elicited T-cell responses in all patients of this trial and in a previous study (9). Besides immunogenicity, initial promising results on the direct clinical activity of neoantigen vaccines are emerging, providing evidence that these therapeutics can yield meaningful clinical bene ﬁt to patients, as recently shown in a ran- domized phase II trial of personalized vaccine mRNA-4157 plus pembrolizumab versus pembrolizumab alone as adjuvant therapy in patients with high-risk melanoma (29). These recent results also highlight the potential of a personalized vaccine in earlier-stage disease, particularly following tumor surgery. Finally, potential biomarkers for lack of clinical response to the combination of vaccine and CPI were also investigated. Tumor immunogenicity is determined not only by the tumor antigenicity itself, but also in ﬂuenced by factors such as the tumor microenviron- ment, including antigen presentation ef ﬁciency. Defects in the APM genes may result in lack of antigen presentation and thereby lack of cytotoxic T-cell-mediated tumor elimination, even in the presence of vaccine-induced T cells. Expression of APM genes was found down- regulated in the PD patients. Tertiary lymphoid structures (TLS) have been shown to play a key role in shaping the immune microenviron- ment of several tumor types, including melanoma (30). Interestingly, our analysis showed a downregulation of chemokine expression necessary for the induction of TLS, such as CCL19, CCL21, and CCR7, in PD patients, that may be relevant for effective treatment outcome, also in the case of combined vaccine and anti-PD-1 treatment. We acknowledge several limitations of our study, in particular the small sample size and the absence of a control arm to estimate the role of vaccine-induced neoantigen-speci ﬁc T cells in determining the disease outcome. Larger cohorts and randomized trials will be neces- sary to establish superior activity of vaccine and anti-PD-1 with anti- PD-1 alone. The expectation is that vaccination will improve the clinical outcome without increasing toxicity, which is in contrast to currently used CPI combinations. Another limitation is the availability of patients ’specimens, either blood or tumors, for deeper character- ization of the immune responses at single-cell resolution by scRNA- seq, and detailed analysis of CD4 and CD8 responses. In summary, our study demonstrated the feasibility of a novel personalized neoantigen-based vaccine that in combination with pembrolizumab was shown to be safe and able to generate tumor speciﬁc T cells capable of traf ﬁcking to the tumor. The data presented here support the use of this vaccine platform as a valuable therapeutic option with a potential to be used in a wide range of disease settings and indications.Authors ’Disclosures A. D ’Alise reports employment with Nous com Srl.