Eligible patients were aged 18 years or older; with advanced pancreatic cancer confirmed histologically or cytologically, chemotherapy intolerance or disease progression after second-line treatments, at least one measurable lesion as per investigator-assessed Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1), an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and good functioning major organs, such as heart, liver and kidney. All patients could provide sufficient tumor tissue and blood samples for whole exome sequencing (WES) and RNA sequencing (if fresh tumor tissue is available).
Key exclusion criteria included: having other malignant tumor except for cured basal cell carcinoma, thyroid carcinoma, or cervical dysplasia; lacking identified neoantigen by sequencing; having received bone marrow or stem cell transplant; allergic to any drugs, polypeptides, or other potential immunotherapies.
Trial design and treatment
This was a single-arm, open-label, investigator-initiated clinical study at Zhejiang Provincial People’s Hospital in China (NCT03645148), with the primary endpoints of safety and feasibility, and secondary endpoints of efficacy evaluated through progression-free survival (PFS), overall survival (OS) and neoantigen-specific immune responses. The safety of the study was assessed on the basis of occurrence of adverse events (AEs). The feasibility of this trial was assessed by whether the neoantigens could be identified by our in-house pipeline iNeo-Suite and the synthesis of the peptide vaccines could be accomplished for clinical use.
iNeo-Vac-P01 for each patient comprises 5~20 peptides at lengths of 15~35 amino acids. The peptides were firstly grouped into 2~4 pools based on their HLA typing, affinity and allele frequency, and then injected subcutaneously (s.c.) in the upper arms and paraumbilical region respectively, at the dose of 100 μg per peptide. Thirty minutes prior to each injection of iNeo-Vac-P01, 40 μg granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously nearby the immunization site as adjuvant (10, 12, 19-21). Patients were primed with iNeo-Vac-P01 on day 1, 4, 8, 15 and 22 (prime phase), and boosted with the same vaccine formulations on day 78 and 162 (boost phase). Additional booster shots might be applied depending on the ethics and the patients’ potential benefits from doing so according to the clinical research protocol. Whether or not to apply concomitant medical therapies such as chemotherapy and immune checkpoint blockade was decided by clinicians according to each patient’s clinical response. The whole treatment regimen of each patient was summarized in Table S1.
Clinical assessment, monitoring and follow-up of patients with advanced pancreatic cancer in this study were conducted, including physical examination such as ECOG performance, vital sign, blood test and urinalysis for the safety evaluation; imaging examination at baseline and post-vaccination for clinical efficacy assessment; IFN-γ Enzyme-Linked Immunospot (ELISpot) Assay and flow cytometry (T cell subsets and cytokines) applied pre- and post-vaccination for the detection of specific immune response.
Tumors were assessed by investigators according to RECIST v1.1 criterion at baseline and approximately every 8 weeks thereafter. The clinical response of each patient was evaluated not only throughout, but also at a regular interval of 3 months after the whole vaccination regimen until disease progression, development of cumulative toxic effects or patient discontinuation. The occurrence and severity of adverse events (AEs) were recorded, and graded based on the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0) throughout the treatment period.
The study protocol was approved by the Institutional Review Board and Independent Ethics Committee, and implemented in accordance with the Declaration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice. All patients had signed informed consent forms before immunization.
Generation of personalized peptide neoantigen vaccines
To identify mutation-derived neoantigens, tumor tissues and blood samples were obtained from patients either directly after surgery, or by biopsy or intravenous blood sampling. Whole exome sequencing (WES) was conducted on these samples using Hiseq 4000 NGS platforms (Illumina) with coverage depths of 500x for tumor cells and 100x for blood cells (Novogene Biotech Co., Ltd., Beijing, China)(22-26). In particular, formalin-fixed paraffin-embedded (FFPE) samples were used for WES when fresh tumor samples were unavailable.
The bioinformatics analysis was performed by our in-house pipeline iNeo-Suite consisting of multiple modules: sequencing read filtering, genome alignment, mutation calling, HLA typing, MHC affinity prediction, gene expression profiling, vaccine peptide sequence design, and mutation centered prioritization based on therapeutic potency (Supplementary Methods).
To generate personalized peptide neoantigen cancer vaccine, iNeo-Vac-P01, customized clinical-grade long peptides were manufactured through chemical synthesis at GMP-like standard (bacteria-free, > 95.0% purity, and quantities of bacterial endotoxin less than 10 EU/mg). The water solubility of these synthesized peptides was tested before excluding insoluble peptides from iNeo-Vac-P01 formulation.
IFN-γ Enzyme-Linked Immunospot (ELISpot) Assay
To confirm the immunogenicity of iNeo-Vac-P01, ELISpot assays were performed for each patient at a series of timepoints pre- and post-vaccination. Peripheral blood (10-30 mL) was obtained from each patient for the isolation of peripheral blood mononuclear cells (PBMCs). PBMCs were then co-incubated (2×105 cells per well) with peptides for 16-24 hours using Human IFN-γ pre-coated ELISpot kit following the standard protocol. An automatic plate reader with appropriate parameters was used to count spots in ELISpot plate (Supplementary Methods).
T cell receptor (TCR) sequencing
To monitor the change of T cell population of each patient, T cell receptor (TCR) β chains were sequenced before and after vaccination. RNA extraction of PBMCs was performed using RNeasy Plus Mini Kit (Qiagen). Samples were analyzed by High-throughput sequencing of TCR using ImmuHub TCR profiling system at a deep level (ImmuQuad Biotech). Briefly, a 5’ RACE unbiased amplification protocol was used. Unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis were used to control bottlenecks and eliminate the errors of PCR and sequencing. Sequencing was performed on an Illumina HiSeq system with PE150 mode (Illumina). One common adaptor with UMI was added to the 5’ of cDNA during the synthesis of first-strand cDNA. One reverse primer corresponding to the constant (C) regions of each TCRα and β was designed to facilitate PCR amplification of cDNA sequences in a less biased manner. The UMIs attached to each raw sequence reads were applied for sequencing error correction and PCR duplication removal. V, D, J and C segments were mapped with IMGT. CDR3 regions were extracted, and clonotype assembled for all clones. The special nucleotide/amino acid sequences of CDR3 region of TCRβ subunit were determined. Those with out-of-frame or stop codon sequences were removed from the identified TCRβ repertoire. We further defined the amount of each TCRβ clonotype as the total number of TCRβ clones sharing the same nucleotide sequence of CDR3 region.
Cytometric analysis of T-lymphocyte and cytometric bead array (CBA) analysis of cytokines
To quantify the activation of T cells after vaccination, flow cytometry was applied to analyze the proportions of different types of T cells extracted from patients’ peripheral blood samples and labeled with several antibodies. To examine the cytokines secreted from activated T cells after vaccination, the concentrations of cytokines in patients’ peripheral blood were measured by CBA, according to the manufacturer’s protocol (Supplementary Methods).
Data from the patients received at least one dose of iNeo-Vac-P01 was analyzed for the assessment of safety and clinical efficacy. Descriptive statistics was applied to determine the characteristics of baseline, and assess the safety of iNeo-Vac-P01. The target lesions of each patient were measured before the treatment and then every two months during the treatment to monitor the changes in lesion sizes. Disease control rate (DCR) was defined as the proportion of patients who had complete response (CR), partial response (PR) and stable disease (SD) for best clinical response. Standard RECISTv1.1 guideline was applied for the analyses of all clinical data. The survival curves were plotted by GraphPad Prism 5 (v5.01).