DNA and RNA preparation
Tumor tissues were obtained by fiber bronchoscopy biopsy, supraclavicular metastatic lymph node resection, fine needle aspiration of metastatic lumbar lesion and fine needle aspiration of lung tumor lesions and stored with 1 ml of RNA later (Thermo Fisher, #AM7020). Blood was collected and mixed with an EDTA- based anticoagulant. DNA and RNA were extracted from the same tumor tissue with the All Prep DNA/RNA Mini Kit, and DNA from EDTA-anti coagulated peripheral blood was extracted with the QIAamp DNA Blood Mini Kit. Quality of DNA/ RNA was evaluated by 2100 Bioanalyzer. Trio samples (two DNA samples from tumor and blood, and one RNA sample from only the tumor) for each patient were prepared for the subsequent sequencing process.
Peptide synthesis. The peptides were 5–15 amino acids in length; peptides were synthesized by standard solid-phase synthetic peptide chemistry and purified using reversed-phase high- performance liquid chromatography (HPLC). The type of HPLC used was LC-20AT. The experimental conditions were Pump A: 0.1% trifluoroacetic in 100% water; Pump B: 0.1% trifluoroacetic in 100% acetonitrile; total flow: 1ml/min; wave- length: 214 nm; analytical column type: SHIMADZU Inertsil ODS-SP (4.6×250mm×5um).
PBMCs were isolated from patients’ peripheral blood. 5–10 × 106/ml elutriated PBMCs were inoculated into T175 flasks containing AIM-V medium and incubated for 3 hat 37 °C and 5% CO2. Then, the suspended cells were collected and frozen in liquid nitrogen. The adherent cells were washed and cultured in AIM-V medium containing 1% autologous serum, clinical grade human GM-CSF (1000 IU/ml) and animal-free research grade IL-4 (500 IU/ml). After 5 days, the neoantigen peptides (50 μg/ml) were added to the immature DCs. Following 20–24 hours peptide loading, DCs were matured with TNF-α (10 ng/ml), IL-1β (10 ng/m), IFN-γ (1000 U/ml), prostaglandin E2 (PGE2, 250ng/ml) R848 (1μg/ml), and polyinosinic- polycytidylic acid (20 ng/ml; poly (I:C)) and incubated for 20–24 hours.
Patients were pretreated with cyclophosphamide at a dose of 250 mg/m2 1 day before injection of Neo- DCVac. The prepared Neo-DCVac was vaccinated subcutaneously at both axillary and inguen regions bilaterally at day 0. GM-CSF was administered at a dose of 0.075 mg during the following 5 days (days 1– 5).
AEs and Toxicity
All adverse events were limited to grade 1 or 2. All patients experienced grade 1 skin injection-site reactions. Patient 4 developed transient grade 1 neutropenia after receiving Neo-DCVac treatment, which was relieved without any treatment. Patient 12 developed a grade 2 itchy rash throughout his trunk and extremities. No other specific treatment was administered, and his rash waned after vaccination ceased. Neo-DCVac did not increase the risk of immune-related adverse events related to ICIs. No toxicity was dose-limiting or resulted in dose delay or treatment discontinuation.
The median progression-free survival (PFS) was 5.5 months (95% confidence interval (CI, 1.9–9.2), and the median overall survival (OS) was 7.9 months (95% CI, 5.9–10.0). Three (25%) of 12 patients achieved an objective response. All of the responses were PR, with no complete responses (CRs) recorded. Six (50%) of 12 patients experienced a decrease in the size of their target lesions, nine (75%) of 12 patients achieved disease control, and progressive disease (PD) was recorded in three patients (25%).
This findings from this pilot study have proven that Neo- DCVac is feasible, safe, and capable of eliciting specific T-cell immunity and therapeutic benefit. The first findings for the activity of a neoantigen-based DC vaccine treatment in patients with advanced NSCLC. Perhaps of greatest importance, Neo-DCVac could have implications in a wide range of cancers.
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Scientific article publishing date 1/21/2021
Immucura identifier BSC21_292EN