Trial participants
Table of Contents
- Trial participants
- Trial design, treatment and oversight
- Peptides
- IFNγ ELISpot assay
- Characterization of CD4⁺ and CD8⁺ T cell responses
- In vitro stimulation and functional validation of CDC7 neoAg contained in Nous-209
- Genomic DNA extraction and whole-exome sequencing (WES)
- Existing WES data processing
- Personalized Cancer Vaccine Trials: A Focus on Immunogenicity
Our study population comprised individuals aged 18 years or older with a diagnosis of LS,as determined by documented carrier status of a deleterious or pathogenic or suspected to be deleterious or pathogenic (known or predicted to be detrimental or result in loss of function,respectively) germline mutation in MLH1,MSH2/EPCAM,MSH6 or PMS2,identified by a Clinical Laboratory Improvement Amendments-approved laboratory test. Consistent with the primary objectives to evaluate the safety, tolerability and immunogenicity of Nous-209 amongst healthy LS carriers, eligible trial participants had no evidence of active or recurrent invasive cancers for at least 6 months before screening and received no cancer-directed treatment (surgery, systemic therapy, hormonal therapy or radiation) within 6 months before screening.We excluded participants who had histologic evidence of high-grade dysplasia and/or invasive cancer at baseline screening. Eligible participants had adequate organ function and Eastern Cooperative Oncology Group (ECOG) performance status 0-1. Except for cardiopreventive aspirin (<100 mg daily), participants consented to refrain from the use of aspirin, nonsteroidal anti-inflammatory drugs or cyclooxygenase inhibitors for the duration of the study treatment. At the study entry, a total of nine participants reported taking aspirin at a low dose (<81 mg orally daily) for cardiovascular or cancer prevention. Three of them decided to discontinue its use upon recruitment and six continued,with three stating the use for cardiovascular and three stating the use for cancer-preventive reasons. Participants consented to refrain from receiving other vaccinations within the first 10 weeks of initiating study treatment and from receiving adenoviral-based vaccines for the duration of study participation (including postintervention follow-up from week 9 through week 52). We excluded individuals with active infection, including human immunodeficiency virus, hepatitis B virus (HBV) or hepatitis C virus (HCV) except those with documented laboratory evidence of cleared HBV or HCV infection, individuals with a history of organ allograft or other history of immunodeficiency or individuals with a intercurrent condition requiring systemic treatment with corticosteroids (>10 mg daily of prednisone equivalents) or other immunosuppressive medications within 14 days of study treatment. Females who were pregnant or breastfeeding or planning to become pregnant or men attempting or planning to conceive children within 6 months of the end of study treatment were excluded. Detailed inclusion and exclusion criteria are available in the study protocol (Supplementary Information).
Trial design, treatment and oversight
The trial was a phase 1b/2 single-arm, open-label, multicenter, prospective study originally designed with the coprimary endpoints of safety and immunogenicity following initial vaccination with Nous-209 monotherapy. To achieve a goal of at least 36 individuals evaluable for the primary immunogenicity endpoint, up to 45 participants were enrolled between November 2022 and November 2023 at four institutions (The University of Texas MD Anderson Cancer Center (MDACC), the University of Puerto Rico, Fox Chase Cancer Center and City of Hope) within the National Cancer Institute (NCI) iCAN PREVENT clinical trial consortium. at baseline, all participants underwent standard-of-care screening lower endoscopy (flexible sigmoidoscopy or colonoscopy). Confirmed eligible participants received initial Nous-209 vaccination as a single 1-ml IM injection of GAd20-209-FSPs (nominal concentration of 2 × 1011 viral particles per ml) at week 0 (prime), followed by a single 1-ml IM injection of MVA-209-FSPs (nominal concentration of 2 × 108 infectious units per ml) at week 8 (bo
from blood collection. pbmcs were isolated using Leucosep Bio-one polypropylene tubes (prefilled; Greiner, Merck) following the manufacturer’s instructions. Cryopreserved cells were thawed,washed,counted and rested overnight before use in immunological assays.
Peptides
A set of 976 recombinant, lyophilized peptides, with the majority of them being 15 aa in length, overlapping by 11 aa and spanning the entire sequence of Nous-209, were produced by JPT Peptide Technologies. Individual FSPs were covered by its specific pool of overlapping peptides and than arranged in 16 peptide pools for immunogenicity assessment, as described below. Lyophilized peptides were reconstituted at 40 mg ml−1 in sterile DMSO (Sigma, D2650), aliquoted and stored at −80 °C. To prepare pools 1-16, the peptides were mixed to a final concentration of 0.4 mg ml−1 for each peptide.
IFNγ ELISpot assay
IFNγ ELISpot assays were performed ex vivo in triplicate with 2 × 105 PBMCs per well in R10.PBMCs were resuspended in R10 medium, stimulated with a set of peptides designed to cover the 209 FSPs encoded by the vaccine and arranged into 16 peptide pools (P1-P16) at a final concentration of 3 µg ml−1. Cells were plated in ELISpot plates (human IFNγ ELISpot PLUS kit, Mabtech) and incubated for 18-20 h at 37 °C in a humidified CO2 incubator. At the end of incubation, the ELISpot assay was developed according to the manufacturer’s instructions. Spontaneous cytokine production (background) was measured by incubating PBMCs with medium alone, supplemented with the peptide diluent DMSO (negative control, Sigma-Aldrich), whereas CEFX (JPT Peptide Technologies), a pool of known peptide epitopes for a range of HLA subtypes and different infectious agents, was used as positive control.Results are expressed as SFCs per 106 PBMCs in stimulated cultures after subtracting the DMSO background. A response was considered positive if (1) the number of SFCs per 106 PBMCs ≥ 50 and (2) it was at least twice the DMSO background value. A subject was classified as a responder if reactivity to at least one of the 16 FSP peptide pools is induced after vaccination. If a subject exhibited preexisting reactivity to a peptide pool at baseline, the vaccine was expected to enhance this response by at least 80% in at least one of the 16 FSP peptide pools; in this case, such participants were considered as responders. NeoAg vaccine peptide pools were deconvoluted to identify immunogenic peptides by IFNγ ELISpot assays ex vivo or after in vitro stimulation.ELISpot plates were analyzed on the CTL ImmunoSpot S6 worldwide analyzer.
Characterization of CD4⁺ and CD8⁺ T cell responses
To characterize neoAg-induced CD4⁺ and CD8⁺ T cell responses, CD8+ T cells were selectively depleted from the total using anti-CD8 microbeads (Miltenyi Biotech, 130-045-201) following the manufacturer’s instructions. The CD8− cell population was then stimulated with either individual peptides or peptide pools (final concentration 3 μg ml−1) and T cell responses against specific peptides were assessed using an IFNγ ELISpot assay. The depl
7 The codon-optimized CDC7 50-mer construct was cloned into a lentiviral backbone obtained from vectorbuilder (plasmid VB240607-1533chz). The design of the construct included an N-terminal signal peptide (MSPMRVTAPRTLILLLSGALALTETWAGS), the mutated CDC7 epitope containing the HLA-A*03:01-restricted 15-mer (TSRILNLQVLKKILR) with its minimal 9-mer core (TSRILNLQV), an MHC I trafficking domain (MCLRLRTKLEKALSALFIWPQHSYKIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGG) and a flexible C-terminal linker (KGGSYSQAASSDSAQGSDVSLTA). This configuration ensured efficient routing of the CDC7 construct through the secretory pathway and enhanced antigen processing and presentation for recognition by neoAg-specific T cells. HCT116 cells were transfected to express HLA-A11:02, A24:01, A03:01 and B07:02, along with their endogenous alleles (HLA-A02:01, A01:01, and B45:01). The transfection process followed a two-step protocol. In the first step, HLA allele expression cassettes were cloned into PiggyBac transposon vectors under constitutive promoters and cotransfected with a transposase helper plasmid into HCT116 cells using Lipofectamine 3000 according to the manufacturer’s instructions. The fluorescent reporters (red fluorescent protein, blue fluorescent protein and enhanced green fluorescent protein (EGFP)) were included to enable tracking of transgene expression and selection of stable clones by drug selection, followed by flow cytometry sorting to ensure the expression of all transgenes within single cells. In the second step, HCT116 cells carrying the HLA constructs were transduced with ready-to-use lentiviral particles generated by VectorBuilder carrying the CDC7 50-mer construct. Cells were plated 1 day before infection, exposed to viral supernatant supplemented with 8 μg ml−1 polybrene and spinoculated at 800g for 90 min at 32 °C to enhance transduction efficiency. After overnight incubation, the medium was replaced with fresh complete DMEM and cells were expanded for 5-7 days. Puromycin selection was applied to enrich for stable integrants, which were further purified by FACS on the basis of EGFP expression. Stable clones were later validated by PCR and sequencing for integration, by western blot and flow cytometry for expression and by functional assays to confirm HLA surface expression and CDC7 MG presentation.
In vitro stimulation and functional validation of CDC7 neoAg contained in Nous-209
For the functional evaluation of neoAg-specific immune responses, the 15-mer peptide CDC7 (TSRILNLQVLKKILR), which is restricted to MHC I, was used for in vitro validation. The peptide was synthesized by JPT Peptide Technologies and used to stimulate pbmcs derived from LS trial participant 12 collected at baseline and at week 8 after Nous-209.PBMCs were cultured in R10 medium (RPMI 1640 with L-glutamine (Corning,10040CV),10% heat-inactivated FBS (HyClone,SH30070.03), 10 mM HEPES buffer (Corning, 25060-CI) and 1× penicillin-streptomycin (Corning, 30002CI)), supplemented with 330 U per ml recombinant human IL-7. Stimulation was carried out using 4 µg ml−1 CDC7 peptide per well, wit
Luminescence was recorded on a plate luminometer, normalized to control wells and expressed as percentage apoptosis relative to baseline.
Genomic DNA extraction and whole-exome sequencing (WES)
Genomic DNA was extracted from five serial slides from formalin-fixed paraffin-embedded (FFPE) blocks of endoscopic resections of colorectal adenomas from on-study colonoscopies. First, we deparaffinized the tissue sections with xylene and 100% alcohol. then, tissues were collected and incubated with lysis buffer in the presence of proteinase K using the Roche microRNA isolation kit. Lysates were centrifuged for 30 min at 4 °C at 15,000 rpm and cell pellets were used for extraction of genomic DNA using the AllPrep DNA/RNA FFPE kit (Qiagen), following the manufacturer’s protocol. DNA quality was assessed using TapeStation analyzer; then, Twist exome capture, library preparation and raw sequencing were performed by the Advanced Technology Genomics Core at The university of Texas MDACC using the Illumina NovaSeqX platform. Alignment of WES data was performed using BWA-mem (version 0.7.19) with default parameters to human genome reference hg38. Duplicate reads were marked with GATK (version 4.6.2.0). Base quality recalibration was performed with GATK Apply BQSR.
Existing WES data processing
Tumor exome data were processed starting from the raw data (FASTQ files), which were downloaded from the National Center for Biotechnology Information under BioProject PRJNA954699. A preliminary quality control of the raw sequence data was performed by filtering out reads of low quality with Trimmomatic (version 0.33)33.The remaining reads were aligned on the GRCh37 human genome BWA-mem (version 0.7.17-r1188)34. Multimapping reads were filtered out using SAMtools (version 1.9)35. Optical duplicates were marked using Picard’s MarkDuplicates tool with Picard tools. DNA alignments were further optimized at regions around indels and base scores were recalibrated
Personalized Cancer Vaccine Trials: A Focus on Immunogenicity
Researchers are increasingly employing personalized cancer vaccines, designed to trigger an immune response against unique mutations within a patient’s tumor. These vaccines prioritize peptides predicted to bind strongly to MHC class I molecules, aiming to maximize the activation of cytotoxic T lymphocytes. Clinical trials,like those utilizing Simon’s minimax two-stage design,assess immunogenicity – the ability to provoke an immune response – through assays such as ELISpot.
Neoantigen Identification and Peptide Prioritization
Personalized cancer vaccines rely on identifying neoantigens, which are tumor-specific mutations that the immune system can recognize as foreign. The process begins with genomic sequencing of the patient’s tumor and normal tissue to pinpoint these mutations. Subsequently,researchers predict which mutated peptides will bind with high affinity to the patient’s MHC class I molecules,which present antigens to T cells.
Peptides are prioritized based on these MHC class I binding affinity predictions. This selection process is crucial because only peptides that effectively bind to MHC class I molecules can be presented to T cells and initiate an immune response. A study by roudko et al. (2020) highlighted shared immunogenic poly-epitope frameshift mutations in microsatellite unstable tumors, providing a foundation for understanding immunogenicity in cancer vaccine advancement. Reference
Statistical Design of Immunogenicity Trials
Clinical trials evaluating personalized cancer vaccines frequently enough employ a Simon’s minimax two-stage design to efficiently assess immunogenicity. This design allows for early termination of the trial if the vaccine demonstrates insufficient efficacy or, conversely, allows for continued enrollment if promising results emerge.
In this design, immunogenicity is defined as the proportion of treated participants exhibiting an immune response, as measured by assays like ELISpot. The primary efficacy endpoint is evaluated based on prior evidence, as demonstrated in the Roudko et al. (2020) study,which informs the expected response rate. Reference
elispot Assay for Measuring Immune Response
The ELISpot (enzyme-linked immunosorbent spot) assay is a commonly used immunological technique to measure the number of cells that secrete specific cytokines, such as interferon-gamma, in response to stimulation with vaccine peptides. This assay provides a quantitative assessment of the cellular immune response elicited by the personalized cancer vaccine.
A positive ELISpot result indicates that the vaccine has successfully stimulated T cells to recognize and respond to tumor-associated antigens. The number of spot-forming cells directly correlates with the magnitude of the immune response. This data is critical for determining the immunogenicity of the vaccine and guiding further development efforts.
