Study design and patients
Between June 2017 to June 2020,Patients aged between 18 and 75 years were provided informed consent and enrolled to a prospective,single center, single-arm,pilot clinical trial (NCT03205930) approved by the Ethics Committee of Lianyungang Clinical College of Nanjing Medical University (2017008). The study was conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent.
Eligible patients were clinically or radiographically evident, pathologically confirmed stage IV or recurrent metastatic NSCLC, including squamous cell carcinoma and adenocarcinoma, who failed multiple lines of therapy(at least two systemic therapies).Other inclusion criteria included a life expectancy of over 3 months,an Eastern Cooperative Oncology Group (ECOG) score of 0–2,adequate hematologic and organ functions,tumor lesions could be evaluated by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST) ,cessation of any cancer therapy for at least 1 month before enrollment.Key exclusion criteria were previous treatment with anti-CTLA-4 or anti-PD-1/PD-L1 therapy, active autoimmune disease.The patients enrolled were undergoing tumor biopsies and blood withdrawing, including formalin-fixed paraffin-embedded (FFPE) samples, biopsy specimen, serum samples and serous effusions.
Study treatment
All participants were treated with a subcutaneous injection of 1–10×107 mature DCs (mDCs) loaded with neoantigens and multiple antigens on Day 8 and autologous cytotoxic T lymphocytes (CTL) induced by mDCs on Day 27 (1–10 × 109 per infusion cycle). A treatment cycle was defined as 28 days (4 weeks). The enrolled patients received at least 3 cycles,up to 12 cycles. Patients with disease progression (PD) ,death or unmanageable toxic effects were taken off of the study(Fig. 1).
Study endpoints and assessments
The primary endpoints of this study included safety and feasibility. The secondary endpoints included progression-free survival (PFS,)overall survival (OS), objective response rate (ORR), and disease control rate (DCR).
The tumor assessment of each patient, including contrast-enhanced computed tomography and magnetic resonance imaging (MRI) of the brain,was conducted before the first treatment. Tumor responses were assessed using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 criteria after 3 cycles and subsequently every 12 weeks based on investigator assessment. Toxicity assessments, adverse events, laboratory values, ECG, and vital signs were assessed regularly and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (v.4.03) for 36 months after the final cycle or until the patient was lost to follow-up.
Whole Exome Sequencing
FFPE tumor samples and matched autologous peripheral blood mononuclear cells (PBMCs) samples of each patient were sent to the facility of Geneseeq Technology Inc (Nanjing, China) for subsequently whole exome sequencing( WES) analysis[13–14].
Bioinformatics and mutation calling
All raw data output from sequencing were filtered out adapter and trimming low quality reads with Illumina illustration. Clean reads were aligned to the reference genome hg19 using BWA. Duplicate site were makered using Picard. The paired tumor and normal data were used to detected single nucleotide variations (SNV) and small insertions/deletions (Indel) using Mutect2. All detected variation were annotated by Annovar and filtered with frequence more than 5% and over than 20 supported reads for further analysis.
Human leukocyte antigen (HLA) typing
HLA class I major loci A,B,C were typed at four-digit resolution by Polysolver[15]using normal sample’s WES sequence data.
Neoantigenic peptides(neo-peptides) prediction and prioritization
All mutation were annotated by Annovar. NeoORF were annotated manually and translated into amino acid sequence. The 31 long peptides which containing mutation in middle site and extend 15 amino acids of both sides were selected for epitopes prediction. All possible 8-11mers peptides sequences were computed affinity and stability cooperated with the patient’s HLA-I subtypes using netMHC4.0/netMHCpan3.0 and netMHCstab/netMHCpan 2.0.
Epitopes with stability more than 2 hours were defined as positive presented epitopes. Neo-peptide which contained 31 amino acids were classified as four levels: Neo-peptides with more than two positive epitopes were defined as level 4.Otherwise, neo-peptides containing only single epitope with affinity value lower than 150nM were defined as level 3, and neo-peptides containing single epitope affinity value which ranged from 150nM to 500nM were defined as level 2, and affinity value higher than 500nM were defined as level1. All neo-peptides were ranked by high levels and within same level ranked by the mutation frequence. For each patient, the top 1–15 neo-peptides were selected for further peptides synthesis and using in further aNASCT cell culture.
Generation and assessment of aNASCT
MASCT is the first therapeutic intervention combining DCs vaccines and ACT in a single treatment modality to elicit both active and passive immune response, MASCT was prepared as our published study[7]. Peripheral blood mononuclear cells (PBMCs) from patients were obtained by density gradient centrifugation using Lymphoprep (Nycome-dPharma, Oslo, Norway) and incubated 1.5 h at 37°C in a saturated 5% CO2 incubator. After that, adherent monocytes were cultured with GM-CSF (1000 U/mL) and IL-4 (500 U/mL) in AIM-V (Gibco, Carlsbad, CA) to differentiate into immature DCs (imDCs). The imDCs were pulsed by a peptide pool of neo-antigens and basic antigens (1 µg/mL/peptide) and then cultured with a maturing cocktail (IL-6, 1000 U/mL; TNF-α, 1000 U/mL; IL-1β, 10,000 U/mL; PEG2, 1 µg/mL; Poly I:C, 10 µg/mL) to differentiate into antigen-presenting mDCs. In order to prepare for the activated CTL infusion, frozen non-adherent PBMCs were co-cultured with antigen-loaded mDCs in the presence of IL-2 (1000 U/mL; R&D Systems Inc, Minneapolis, MN) about 4 weeks .The anti-CD3 antibody (50 ng/mL; eBioscience, Inc., San Diego, CA) was added 3 days after co-culturing. The autologous T cells of Neo-ASCT were then incubated with 1.5 mg SHR-1210 (a fully human IgG4 monoclonal antibody against PD-1) [16]ex vivo for 40 min in a 37°C thermostat, referred to as aNASCT, and finally transferred to patients.
Flow cytometry analysis
The circulating tregs from patients were assessed using flow cytometry as previously described[7]. Antibodies for surface markers and intracellular protein staining were obtained from BD Biosciences including anti-CD80, -CD83, -CD86, -CD3, -CD4, -CD8, -CD25, -CD127, -PD-1, -IL-2,IL-4,IL-6,IL-10,TNF- α,-IFNγ. All the flow cytometry assays were determined using a FACSCalibur Flow Cytometer (BD Pharmingen), and the data were analyzed by the FlowJo software (Tree Star Inc.).
IFN-γ Enzyme-Linked Immunospot Assay (ELISPOT)
IFN-γ ELISPOT kit (U-CyTech Biosciences, Netherlands) was used to detect the levels of peptide reactivity in the CTL. The ELISPOT assay was conducted and analyzed according to standard protocol. About 2×105 cells were incubated with the neoantigen peptides pool,positive peptides or irrelevant peptides using a 48-well ELISPOT plate overnight at 37°C in a saturated 5% CO2. The resulting spots were scanned by Elispot CTL Reader (Cell Technology Inc, Columbia, MD) and analyzed with Elispot software (AID, Strassberg, Germany).
Statistical Analysis
Patient characteristics, clinical outcomes were presented using simple descriptive statistics. GraphPad Prism 9.0 software was used to plot survival curves and perform data analyses. Data samples were compared using a 2-tailed t-test.The Kaplan-Meier curves was used to estimated PFS and OS with corresponding 95% CIs. P < 0.05 was considered significant in all the analyses.