D ’Alise and G. Leoni contributed equally as the co- ﬁrst authors to this article. E. Scarselli and O. Bechter contributed equally as the co-senior authors to this article. Corresponding Author: Anna Morena D ’Alise, Immunology, Nouscom, Rome, Italy. E-mail: m.dalise@nouscom.com Clin Cancer Res 2024;30:2412 –23 doi: 10.1158/1078-0432.CCR-23-3940 This open access article is distributed under the Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license. /C2112024 The Authors; Published by the American Association for Cancer Research AACRJournals.org | 2412Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 neoantigens simultaneously, with the potential advantage of covering heterogeneity and curtailing tumor immune escape. Preclinical studies in various murine tumor models have demonstrated the synergy between anti-PD-1 therapy and viral vectored vaccines encoding tumor neoantigens, to eradicate large established tumors (8 –10). Recently, vaccination based on GAd and MVA vectors has been also employed to target shared neoantigens in metastatic patients with mismatch repair de ﬁciency/microsatellite instability (dMMR/MSI; ref. 9). More speciﬁcally, results from a phase I trial combining anti-PD-1 antibody with GAd20 and MVA encoding shared tumor neoantigens in pati- ents with metastatic colorectal cancer, gastric, and gastro-esophageal MSI tumors have been recently published. The results of the trial demonstrated that the combination treatment was safe, highly immu- nogenic, and showed promising early signs of clinical ef ﬁcacy (9). Here we report translational results from a phase Ib, ﬁrst in human trial (NCT04990479) in patients with metastatic malignant melanoma. The study evaluated feasibility, safety, immunogenicity, and antitumor activity of NOUS-PEV PEV in combination with the PD-1 blocking antibody pembrolizumab. We examined the induction of neoantigen- speciﬁc T-cell responses before and after vaccination, and showed expansion after treatment of vaccine-induced T-cell clonotypes rec- ognizing neoantigens in the tumors of vaccinated patients. Moreover, the analysis of transcriptomic data obtained from pretreatment biop- sies allowed the identi ﬁcation of signatures potentially associated with resistance to the combo treatment. Overall, these results support the potential of this personalized viral-based prime/boost vaccine as a promising immunotherapeutic approach to enhance antitumor responses and the use of biomarkers to inform on the responsiveness to combined vaccine and anti-PD-1 treatment. Patients and Methods Study design This is a multicenter, open-label, phase I clinical study (NCT04990479), designed to evaluate safety, tolerability, and immu- nogenicity, and to detect any preliminary evidence of antitumor activity of a PEV based on GAd-PEV priming and MVA-PEV boost- ing, combined with standard of care (SoC) ﬁrst-line immunotherapy using an anti-PD-1 checkpoint inhibitor in metastatic non –small cell lung cancer (NSCLC) and unresectable or metastatic melanoma. The representativeness of the study population is described in Supplemen- tary Table S1. The PEV vaccines are prepared on an individual basis, following a tumor biopsy performed at the time of screening and subsequent NGS analysis, to identify patient-speci ﬁc tumor mutations. Both neoantigen-encoding genetic vaccines are administered intra- muscularly using one prime with GAd-PEV and three boosts with MVA –PEV in combination with the licensed programmed death receptor-1 (PD-1)-blocking antibody pembrolizumab in patients. Theﬁrst vaccination with GAd occurs on the day of the 4th administration of pembrolizumab (week 10); MVA is administered as three boosts, every 3 weeks, with the ﬁrst administration 3 weeks post-GAd vaccination. GAd and MVA vaccines are administered at the dose ranges of 5 /C21010to 2/C21011vp and 1 /C2108to 3/C2108ifu, respectively. Further details can be found at https://clinicaltrials.gov/stu dy/NCT04990479. This study was conducted in accordance with ethical principles that have their origin in the Declaration of Helsinki and conducted in adherence to the study Protocol, Good Clinical Practices as de ﬁned in Title 21 of the US Code of Federal Regulations Parts 11, 50, 54, 56, and 312, as well as the International Conference on Harmonization E6 guideline for Good Clinical Practice and applicable regulatory requirements. The study was approved by UZ Leuven Ethics Committee (Study Reference S64578) and by Research Ethics Committee of Fundacion Jimenez Diaz. Written informed consent was obtained from each subject. Vaccine vector generation PEV design was performed according to a pipeline aimed at analy- zing patient next-generation sequencing data collected from tumor and blood to select the best neopeptides suitable for the inclusion in viral vectors. NGS data were produced at CeGAT GBMH. Genomic DNA was fragmented and used for Illumina library construction. Exonic regions were captured in solution using the Twist Human Core Kit. Paired-end sequencing (2 /C2100 bp) was performed with the Nova- seq600 Genome Analyser (Illumina) at a target depth of 30 Gb. RNA was fragmented and the sequencing library was prepared using SMART-Seq mRNA Stranded Kit. Sequencing was performed with the Novaseq6000 Genome Analyser (Illumina) at a target depth of 6 Gb (2 /C2100 bp). The patient HLA typing and the prioritization of somatic variants encoding neopeptides were performed as described previously (11). The transgene design was performed according to a procedure that minimizes the formation of predicted junctional epitopes that may be generated by the juxtaposition of two adjacent neoantigen peptides. A custom tool was developed speci ﬁcally for this purpose. The synthetic cassette aminoacid sequence was ﬁnally retrotranscribed by applying an inverse translation scheme with a minimization of penalty score associated to the presence in the sequence of forward/reverse repeats or 5TNT motifs and homopolymers stretches greater than 6bp. Synthetic transgenes expression cassettes were generated via Gibson assembly (New England Biolabs) of double-strand DNA fragments obtained from different providers (Integrated DNA Technologies, GeneART, Thermo Fisher Scienti ﬁc; Doulix; Of ﬁcinae Bio; Euro ﬁns Genomics); fragments were assembled into acceptor shuttle plasmids containing TetO-CMV promoter (with two Tet Operator repeats) and BGH polyA (GAd-PEV transgenes) or P7.5 promoter (MVA-PEV transgenes). For generation of GAd-PEV vectors, transgene cassettes were transferred into a GAd20 BAC construct by recombineering in SW102 Escherichia coli cells. GAd20 BAC contains the genome of a Great Ape Adenovirus (serotype group C) deleted in E1 and E3 regions, whereas the E4 region is replaced with Ad5 E4 ORF6. Positive clones, selected and characterized by PCR, restriction digestion, and transgene DNA sequencing, were used for vector rescue by transfection in M9 cells [human embryonic kidney (HEK) 293 derivative]. Resulting GAd- PEV vectors were ampli ﬁed in suspension M9 cells and puri ﬁed by anion exchange chromatography (9). The GMP production was conducted by Reithera Sri. For generation of MVA-PEV vectors, transgene cassettes were transferred into MVA Deletion III locus by homologous recombina- tion in AGE1.CR.pIX cells. Recombinant vectors were isolated by aTranslational Relevance This study demonstrated the feasibility of a novel personalized neoantigen-based vaccine that in combination with pembrolizu- mab is safe and able to generate robust tumor-speci ﬁc T cells capable of traf ﬁcking to the tumor. The data presented here support the use of a viral vector-based vaccine platform as a valuable therapeutic option with a potential to be used in a wide range of disease settings and indications, aiming to extend immunotherapy efﬁcacy to patients.NOUS-PEV Vaccine Elicits Potent Antitumor T-Cell Responses AACRJournals.org Clin Cancer Res; 30(11) June 1, 2024 2413Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 combination of marker gene swapping and ﬂorescence-activated cell sorting as described previously (12). A limiting dilution step was performed to ensure clonality of the obtained MVA-PEV vectors, followed by ampli ﬁcation in AGE1.CR.pIX cells. The GMP production was conducted by Reithera Srl. Peripheral blood mononuclear cell preparation Venous blood samples from patients were collected in lithium- heparin Vacutest blood collection tubes (Kima) at different time points and cryopreserved at the clinical sites before shipment to the central lab for immunogenicity assessment. To preserve peripheral blood mono- nuclear cell (PBMC) functionality, isolation and freezing procedures were performed within a maximum of 8 hours from blood collection. The isolation of PBMCs was performed using Leucosep Bio-One Polypropylene Tube (pre ﬁlled; Greiner, Merck) according to the instructions of the manufacturer. After that, PBMCs were counted and frozen with freezing media [10% DMSO (Sigma- Aldrich) and 90% FBS (De ﬁned, HyClone], then placed at /C080/C14C for 1 to 3 days before shipment to the central laboratory. Cryopreserved cells were thawed in thawing media [RPMI1640 (Gibco), CTL wash supplement (Immunospot), 1% L-glutamine (Gibco), and 50 U/ mL of Benzonase (Merck Millipore)]. Cell counts and viability were assessed using the Guava Via Count reagents (Luminex) and module on the Guava Muse Cell Analyzer (Luminex). Ex vivo IFNgELISpot The frequency of IFN gproducing T cells was measured by ex vivo ELISpot-forming cell assay after antigen-speci ﬁc stimulation, as pre- viously described (9). PBMCs were resuspended in R10, stimulated with a set of peptides designed to cover the NOUS-PEV patient speci ﬁc vaccine sequence, and arranged into 6 peptide pools (P1 to P6). Cells were plated at 2 /C2105cells per well in ELISpot plates (Human IFN- g ELISpot PLUS kit, Mabtech) and incubated for 18 to 20 hours in the presence of the peptides in a 37/C14C, humidi ﬁed CO 2incubator. At the end of incubation, the ELISpot assay was developed according to the manufacturer ’s instructions. Spontaneous cytokine production (back- ground) was assessed by incubating PBMCs with the medium only plus the peptide diluent DMSO (negative control) (Sigma-Aldrich), whereas CEFX (JPT), a pool of known peptide epitopes for a range of human leukocyte antigen (HLA) subtypes and different infec- tious agents, was used as positive control. Results are expressed as SFC/106PBMCs in stimulating cultures after subtracting the DMSO (Sigma-Aldrich) background. A response was considered positive if (i) the number of SFC/106cells was greater than 50 and (ii) higher than three times the background DMSO value. A subject is de ﬁned as a responder if reactivity to at least 1 of the peptide pools is induced after vaccination. In vitro expansion of neoantigen-speci ﬁc T cells Forin vitro expansion of antigen-speci ﬁc T cells, PBMCs were cultured in RPMI1640 medium (Gibco) supplemented with L- glutamine (Gibco), HEPES (Gibco), penicillin/streptomycin (Gibco), and 10% heat-inactivated human serum (De ﬁned, HyClone), as described previously (9). Cells (4 /C2106per well) in 1-ml volume were stimulated in a 24-well plate with individual (4 mg/mL) or peptide pool (each peptide at 4 mg/mL) in the presence of IL7 (330 U/mL; PeproTech). On day 3, low-dose IL2 (10 U/mL; PeproTech) was added. Half-medium change and supplementation of IL2 were performed at days 3, 7, and 10. After 12 days, PBMCs were collected and resus- pended in complete medium without cytokines and peptides for overnight resting at 37/C14C. The day after, T-cell response against peptides of interest was tested by IFN gELISpot assay.TCR-seq analysis T-cell receptor (TCR) V bsequencing (TCR-seq) was performed on blood at baseline and post-vaccination, and on IVS expanded T cells. DNA from bulk PBMCs or stimulated speci ﬁc T cells was prepared using a Qiagen DNA Blood and Tissue Extraction Kit according to the manufacturer ’s instructions. Samples were quanti ﬁed using Nanodrop 2000 (Thermo Fisher Scienti ﬁc). DNA was extracted from each col- lected sample and sequenced at CeGaT GMBH. Raw NGS data were analyzed with MiXCR 2.1.11 (13), applying default parameters. The repertoire detected in tumor at baseline and after vaccination was determined was assessed from the RNA-seq data using the MiXCR tool and applying the standard parameters reported in the RNA-seq work ﬂow of the manual. The derived CDR3 sequences were further analyzed by tracking the expression of clonotypes that were shared between pre-/post-vaccination tumor biopsies and in vitro samples stimulated with NeoAg7 minimal 9-mer mutated peptide. Gene expression analysis Differentially expressed genes were estimated by comparing treated versus untreated with a consensus of four different methods: DESeq2 (RRID:SCR_015687; ref. 14), edgeR (RRID:SCR_012802; ref. 15), limma with Voom correction (16), and NOISeq (17). A count matrix reporting the number of reads mapping to each gene was determined by using the Rsubread (PMID: 30783653) package and gene expression was expressed as transcripts per kilobase million (TPM). The Benja- mini –Hochberg correction was applied to the list of differentially expressed genes identi ﬁed by each method. Only genes identi ﬁed by three out of the four methods, with a difference of log2FC of at least /C61 and a corrected Pvalue ≤0.05 were retained. IFNG/IMS signature was computed as described in Cui and colleagues (18); R software was used to implement the signature computation script. Clonality analysis Analysis of clonality of detected mutations was performed by using allele-speci ﬁc copy-number analysis of tumors (ASCAT) software (19) to detect copy-number variation in tumor exome sequencing data, followed by PyClone (20) to estimate the clonality of each somatic mutation, as described previously (9). All the parameters were kept to default except for the input of ASCAT that was created with a custom tool that ﬁlters only the SNPs with at least 30% of mutation allele frequency in blood. The list of ﬁltered SNPs was then passed to the AlleleCounter tool to prepare the input for ASCAT. Data availability Exome and RNA-sequencing (RNA-seq) data generated in this study have been deposited in Sequence Read Archive under the accession code PRJNA1081187. All the other data of this study are available in the article or upon request from the corresponding author. Results Production of GAd and MVA for personalized vaccination is feasible in a short turnaround time This study was open to treatment-na €/C16ve subjects with unresectable stage III/IV cutaneous melanoma and/or PD ligand 1 (PD-L1) ≥50% stage IV non –small cell lung cancer. The study endpoints included the evaluation of safety as the primary endpoint and evaluation of tumor response according to RECIST v1.1 as secondary endpoint. Immu- nogenicity and additional exploratory pharmacodynamics measures and correlates of vaccine-induced immune response were evaluated as exploratory endpoints. As of the data cutoff of June 12, 2023, 7 patientsD’Alise et al. Clin Cancer Res; 30(11) June 1, 2024 CLINICAL CANCER RESEARCH 2414Downloaded from http://aacrjournals.org/clincancerres/article-pdf/30/11/2412/3454679/2412.pdf by guest on 10 December 2024 with unresectable stage III/IV cutaneous melanoma were enrolled in the trial; 6 received the vaccination, whereas 1 patient (Pt 7) progressed prior to any vaccination (demographics and disease characteristics for each patient are shown in Table 1 ). Following pembrolizumab induc- tion treatment (three cycles), NOUS-PEV vaccine was administered intramuscularly with the following scheme: a single priming dose of GAd20 encoding 60 tumor-speci ﬁc nAgs concomitantly with the fourth pembrolizumab infusion at week 10, followed by 3 boosts with MVA, each 3 weeks apart. Baseline and on treatment tumor biopsies were collected at screening and after the ﬁrst MVA administration, respectively ( Fig. 1A ). To produce each personalized neoantigen GAd20 and MVA vac- cine, tumor mutations were ﬁrst identi ﬁed by whole-exome and RNA- sequencing of baseline patient ’s tumor biopsy and matched normal cells from blood, as outlined in Fig. 1A . Neoantigens were then bioinformatically predicted and selected using a previously published algorithm, called VENUS (11). About 60 tumor speci ﬁc neopeptides were selected for inclusion in a codon-optimized arti ﬁcial gene cloned into GAd20 and MVA vectors. Eleven of 12 vaccines were successfully manufactured and released, according to GMP and EU Pharmaco- poeia guidelines, demonstrating the feasibility of this viral vector- based personalized vaccination approach. More speci ﬁcally, the turn- around time (TAT), de ﬁned as the time for vaccine generation and release starting from the day of the tumor biopsy analysis, was an average of 59 days (SD /C64) for GAd20 (expected 56 days), and 81 days (SD/C63) for MVA (expected 81 days; Fig. 1B ).