Data reporting

No statistical methods were employed to predetermine patient sample numbers. The study was not randomized, and some investigators were not blinded during experiments and outcome assessments.

Clinical trial design and treatment

Between November 2016 and December 2018, a single-arm trial was conducted at Tianjin Beichen Hospital in China. Primary endpoints of the trial were safety and tolerability, and feasibility of the personalized approach, and secondary endpoints were antitumor immune reactivity and clinical response. Feasibility of the PPV approach was assessed by whether personalized neoantigens could be successfully identified from patients’ tumor mutational analysis and multiepitope peptide vaccines designed for potential clinical application. All patients provided written informed consent before enrolling in the study.

Patient eligibility

Twenty-nine patients with stage III–IV NSCLC were enrolled in this clinical study of PPV, and 24 were successfully immunized (18 adenocarcinoma and 6 squamous cell carcinoma). The study patients were selected according to the following inclusion criteria: adult patients aged 18 years or more; clinical assessments classified all patients with NSCLC stage III/IV according to NCCN Clinical Practice Guidelines in Oncology, V.3.2016, Non-Small Cell Lung Cancer and the Eighth Edition Lung Cancer Stage Classification; NSCLC diagnosis was confirmed by biopsy and pathological assessment; patients experienced disease recurrence after failing conventional treatments including surgery, chemotherapy, radiotherapy and/or EGFRi therapy, and had no active treatments; patients showed good or moderate Eastern Cooperative Oncology Group performance status (PS≤3); patients were undergoing no other concurrent immunotherapies; pretreatment biopsy samples were available and showed at least one genetic mutation; patients had a life expectancy of >3 months. Patients were excluded if they: were pregnant or lactating; had known or suspected autoimmune disease, or other immune system disease; had systemic cytotoxic chemotherapy or experimental drugs for treatment of metastatic NSCLC within 4 weeks prior to first dose of personalized vaccine (not including EGFRi); had participated in any other clinical trial involving another investigational agent within 4 weeks prior to first dose of personalized vaccine; had liver or kidney dysfunction, severe heart disease, coagulation dysfunction, or hematopoietic impairment; had any active infection requiring systemic treatment; andsuffered from other current malignancies either in progress or treated within the past 5 years. Pretreatment tumor biopsies were required for the trial, and post-treatment biopsies were optional and required additional patient consent. The clinical characteristics of study patients are shown in online supplemental figure S3A, and the EGFRi treatment histories of the 16 EGFR-mutated patients are shown in online supplemental figure S3B.

Generation of personalized neoantigen vaccines

Patient immunizations

Of 29 patients enrolled into the study, three patients passed away while waiting for the vaccine peptides to be produced and were therefore unable to participate. Two additional patients were not treated since their PS increased to 4–5 during production of their vaccine peptides, which then caused them to fail to meet the inclusion criteria of PS<3, and they went on to receive other treatment. Immunizing peptides were synthesized using standard solid-phase synthetic peptide chemistry, purified to >98% using reverse phase high performance liquid chromatography and tested for sterility and the presence of endotoxin to ensure safety and tolerability using methodologies consistent with Good Manufacturing Practice (HengJia Neoantigen Biotechnology (Tianjin)). As shown in online supplemental table S2, 5–14 peptides per patient were synthesized, solubilized individually in sterile phosphate-buffered saline (PBS), and mixed into two separate peptide cocktails, each with two to seven short peptides and one to three long peptides in 1 mL total volume. Peptides binding to the same HLA allotypes were separated into different cocktails to reduce potential antigen competition. Patients received 200 µg of each peptide per immunization, injected subcutaneously into the left and right extremities, and administered weekly for 12 weeks. In order to provide concurrent TLR-7 stimulation of pAPCs, Aldara cream with 5% imiquimod was applied topically as a vaccine adjuvant over the vaccine site immediately after peptide cocktail administration. Patients were permitted to continue immunizations after 12 weeks if desired and in the patient’s best interest. Eleven of the 24 immunized patients continued to receive vaccinations beyond 12 weeks, as shown in online supplemental figure S1 and online supplemental table S1.

Sample collection

Serial PBMC samples were collected at pretreatment, as well as at 4, 8, and 12 weeks postvaccination. A maximum of 15 mL of blood was drawn per month, according to Tianjin Beichen Hospital regulations for patients with advanced-stage cancer. Collection of additional blood samples beyond the 12 weeks of the trial period were optional and required additional patient consent. Extra blood samples were collected from patients 5, 8, and 17, who had all experienced clinical objective responses. Viable PBMCs were collected and stored at −80°C. Collection of postvaccine tumor biopsies was optional but not required in this trial due to the invasive nature of the procedure, uncertain feasibility, and the general reluctance of most patients. Tumor tissues obtained for further bulk T cell receptor Vβ CDR3 sequencing analysis were collected from PPV trial patient 5 after providing written informed consent.

Tumor response evaluation criteria

Objective tumor response assessments were made according to the RECIST (V.1.1) guidelines. We used CT and/or MRI scans to measure selected target lesions (online supplemental table S5). Patients were required to perform at least one pretreatment scan for baseline measurements and another scan at 3–4 months postvaccine for response assessment. Additional patient scans were taken monthly during the first 12 weeks of vaccination if feasible. Target lesions with a minimum size of 10 mm (15 mm for malignant lymph nodes) were measured in the longest diameter by three different radiologists, with the mean of the three independent measurements used for clinical assessments. A maximum of two target lesions per organ were measured, with the two largest lesions selected, up to a maximum of five lesions in total. Tumor burden was calculated as the sum of the diameters of all target lesions (online supplemental figure S4B). Clinical responses were evaluated as follows: CR, complete disappearance of all target lesions; PR, partial response, defined as a 30% decrease in the sum of diameters of target lesions; PD, progressive disease, defined as a minimum 20% increase in the sum of diameters of target lesions or the appearance of new lesions; and SD, stable disease, defined as a change in tumor burden insufficient to qualify for PR or PD. Clinical responses were assessed 3–4 months following the date of the first immunization.

Clinical trial statistical plan

Statistical analysis was primarily descriptive, including enumeration of patients who experienced any adverse events. All statistical tests were two sided with an alpha level of 0.05. CIs to be evaluated were constructed with a significance level of 0.05. Additional exploratory analyses of the data were conducted as deemed appropriate.

ELISA and ELISPOT assays

PBMCs collected prior to the start of immunization and at different time points following PPV were isolated by Ficoll density gradient centrifugation and counted in the presence of trypan blue dye to evaluate viability prior to cryopreservation. For ELISA analysis, 5×10⁵ PBMCs in RPMI-1640 containing 10% fetal bovine serum (FBS) were added to each well of 96-well plate with a total volume of 250 µL. PBMCs were cultured in the presence of vaccine peptide pools, individual vaccine peptides, or irrelevant control peptides (7.5 µg/mL) along with 300 IU/mL interleukin-2 (IL-2) in a 37°C humidified incubator with 5% CO₂ for 5 days. Following 24 hours of peptide restimulation, the IFN-γ concentration of cell supernatants was measured using a human IFN-γ ELISA kit (Dakewe, China), according to the manufacturer’s instructions. A level of IFN-γ secretion twofold or greater over background signal with irrelevant control peptide was considered to be a positive immune response for initial screening.IRC that considered breadth, intensity, and persistence of vaccine-induced immune responses were calculated for each patient as follows: ComboScore=sum of fold increases >2 for all vaccine peptides at all postvaccine time points (4, 8, and 12 weeks). For ELISPOT assays, 2.5×10⁵ PBMCs were prepared in RPMI-1640 containing 10% FBS with a total volume of 150 µL for each well in a 96-well plate. Ex vivo PBMCs were stimulated in triplicate with individual vaccine peptides at a final concentration of 10 µg/mL, and plates were incubated at 37°C in humidified incubator with 5% CO₂ for 36 hours. For patients 11, 12, 16, and 22, in vitro PBMC stimulation was performed for 12 days in the presence of individual (2 µg/mL) or pooled vaccine peptides (2 µg/mL each) and IL-2 (50 IU/mL, Sihuan Biopharmaceutical, Beijing, China), IL-4 (5 ng/mL, Cat. No.: 101–04, PrimeGene), IL-7 (5 ng/mL, Cat. No.:101–07, PrimeGene), and IL-15 (5 ng/mL, Cat. No.:101–15, PrimeGene) prior to performing IFN-γ ELISPOT. Spot detection was performed using a Human IFN-γ ELISpot PRO kit (MABTECH, USA) and normalized to the number of IFN-γ spots detected per 10⁶ PBMC.

Tetramer staining and flow cytometric analyses

Selected custom phycoerethrin (PE) or Allophycocyanin (APC)-conjugated HLA-peptide tetramers (Baylor College of Medicine, USA; MBL, Japan) were successfully generated (online supplemental figure S9A). PBMCs were thawed and resuspended in RPMI-1640 containing 0.5% FBS. One hundred microliters of PBS-1% BSA containing fluorophore-conjugated HLA/peptide tetramer (1:50 dilution) was added to 5×10⁵ PBMCs and incubated at room temperature for 20 min in the dark. Cells were washed with PBS-1% BSA, stained with Fluorescein isothiocyanate (FITC)-conjugated or Phycoerythrin (PE)-conjugated anti-CD8 mAbs (Biolegend, USA, 1:200 dilution), and incubated for 15 min. Cells were then washed and resuspended in 400 µL PBS-1% BSA for flow cytometric analysis (LSRFortessa X-20 Analyzer).

Bulk T cell receptor Vβ CDR3 sequencing analysis

DNA samples were extracted from patient PBMC or pretreatment and post-treatment tumor biopsies from patient 5 using a DNA extraction kit (Qiagen, USA), followed by library construction with two rounds of PCR-based amplification. CDR3 fragments were first amplified using specific primers for each V and J gene, and target fragments of multiplex-PCR products were purified using magnetic beads (A63882, Beckman, Germany). Next, PCR was performed using universal primers, and target fragments 200–350 bp were retrieved and purified by QIAquick Gel Purification Kit (Qiagen, USA). PCR products were then sequenced using the Illumina X10 platform. Single-read CDR3 sequences were eliminated, and the remaining sequences were analyzed to evaluate TCRVβ IMGT clonality of patients before and after treatment, as previously described.53

Single-cell T cell receptor sequencing

HLA-A*1101/KITDFGRAK Tetramer⁺ CD8⁺ T cells from post-PPV PBMCs of patient 5 were sorted using a flow-based cell sorter (BD FACSAria III, USA) and imaged by confocal microscope (Leica SP8, Germany). Sorted Tet⁺ cells were adjusted to 1×10⁶ cells per mL in PBS and loaded on a Chromium Single Cell Controller (10X Genomics, USA) to generate single-cell gel beads in emulsion (GEMs) using a Single Cell 5′ Library and Gel Bead Kit (10X Genomics, USA). Captured cells were lysed, and released RNA was barcoded through reverse transcription to produce barcode-containing cDNA in individual GEMs as per the manufacturer’s introductions. V(D)J sequences were enriched by nested PCR amplification with specific primers targeting conserved TCR sequences. Sequencing was performed on an Illumina NovaSeq platform with 150 bp (PE150) paired ends. Cellranger VDJ was used for analyzing V(D)J recombination, T cell diversity, and pairing of appropriate TCR α and β chain sequences for each individual T cell.

Single-cell sequencing of Tet+ cells resulted in the identification of 639 distinct TCRα/β pairs. However, since tetramer-based cell sorting can result in contamination with non-antigen specific T cells, we chose to select TCR clones for which a minimum of 10 Vβ-CDR3 reads were detected, resulting in 51 unique TCR clones. Four clones with Vβ-CDR3 sequences that showed little or no presence in any PBMC or TIL samples were eliminated from the analysis, and five additional TCR clones with either seven or eight CDR3 reads were included based on their increasing frequencies in PBMC and/or TIL during PPV, as would be expected for a neoantigen-specific T cell clone.

TCR cloning and validation

The variable region sequences of TCR Vα-N1 and Vβ-N1 chains obtained by single cell sequencing were fused to an engineered human constant region to enhance α and β chain pairing.54 These modified Vα and Vβ chain sequences were synthesized and inserted into a EF1a promoter based lentiviral expression vector pCDH to create lentivirus lenti-EF1a-TCR-N1. Healthy donor PBMCs were prepared using lymphocyte separation medium (Stem Cell, Canada). T cells were isolated using the Dynabeads Human T-Expander CD3/CD28 Kit (Thermofisher, USA), mixed with 3 mL X-Vivo15 serum-free medium (Lonza, Switzerland) and cultured at 37°C in 5% CO₂ for 48 hours. Cell density was adjusted to 1×10⁶/mL and cocultured with packaged lentivirus lenti-EF1a-TCR-N1 for 4 days at 37°C in 5% CO₂. TCR-N1 expression and antigen specificity was confirmed by staining with the HLA-A*1101/KITDFGRAK tetramer. A minigene encoding the KITDFGRAK peptide linked to the HLA-A*1101 cDNA through an IRES sequence was synthesized and inserted into lentiviral vector pCDH with EF1a promoter to create lentivirus lenti-EF1a-KIT-A11. Control lentiviral constructs included vectors that expressed either the KITDFGRAK minigene or HLA-A*1101 individually. Lentiviral-transduced A549 lung cancer cells stably expressing the gene(s) of interest were selected through purinomycin-based selection. HLA-A*1101 surface expression was confirmed by staining with an A*1101-specific mAb followed by flow cytometric analysis. Engineered TCR-N1-T cells were cocultured with parental A549, A549-A11, A549-KIT, or A549-A11.KIT cells (20,000 target cells per well) at 37°C in 5% CO₂ with effector-to-target ratios of 1:1, 2.5:1, 5:1 and 10:1. Supernatants were collected after 24 hours of coculture to assess levels of IFN-γ secretion by ELISA. Cytolytic activity of KITDFGRAK-specific TCR transduced T cells also was assessed using a standard chromium-51 (⁵¹Cr) release assay. Target cells were labeled with 100 µL of ⁵¹Cr for 1 hour, then washed and plated at 2000 target cells per well in triplicate. TCR-T cells were incubated for 4 hours at various effector-to-target cell ratios with A*1101-expressing 293 embryonic kidney target cells either transduced to express full-length WT or L858R-mutated EGFR cDNA, or pulsed with WT EGFR peptide KITDFGLAK or L858R mutated KITDFGRAK peptide (5 uM). After the incubation, supernatant was collected from the wells, and ⁵¹Cr was measured with a gamma radiation counter. The percentage of specific target cell lysis was calculated, correcting for background ⁵¹Cr release and relative to a maximum release as indicated by Triton X-100 lysed target cells.

Immunoprecipitation and western blot

H1975 (EGFR-L858R/T790M) and H1299 (EGFR-WT) lung cancer cell lines (ATCC, USA) were treated with different concentrations (0.1, 1, 2, and 5 µM) of EGFRi Osimertinib (LC Laboratories, USA) for different time periods to optimize EGFRi treatment conditions. Cells were washed with cold PBS and lysed using lysis buffer (1% Triton X-100, Sigma, USA) and Halt Protease Inhibitor Cocktail 100X and 0.5 µM EDTA 100X (Thermo Scientific, USA). Lysates were collected and centrifuged at high speed for 30 min at 4°C prior to measuring protein concentration with a Bradford assay kit (BioRad, USA). Preclearing was performed using 10 µL Pierce Protein A/G Ultra Link resin (Thermo Scientific) per sample and incubating for 2 hours at 4°C. Immunoprecipitations were performed using the same amount of total protein and by incubating cell lysates for 18 hours at 4°C with the following antibodies at a 500:1 dilution: antiphospho-EGFR (EMD Millipore), anti-EGFR, Anti-p44/42 MAPK ERK1/2, anti-phospho-p44/42 MAPK ERK1/2, and anti-GAPDH (Cell Signaling Technology). Protein A/G crosslinking beads were added and incubated for 4 hours prior to washing with cold PBS. Samples were run using an SDS-PAGE gradient (8%–16%) gel (Invitrogen, USA). Proteins were transferred to a PVDF membrane (Thermo Scientific, USA), and blots were blocked with 5% milk prior to incubation with specific antibodies overnight at a concentration of 1:1000. Blots were washed and incubated with peroxidase-conjugated antirabbit secondary antibody (1:10,000) (Jackson Immuno Research, USA). Blots were developed using the Super Signal West Pico PLUS Chemiluminescent enhanced horseradish peroxidase substrate (Thermo Scientific, USA) and visualized with X-ray film.

RNA sequencing of lung tumor cell lines

H1975 and H1299 lung cancer cells were treated with 1 µM of EGFRi Osimertinib (LC Laboratories, USA) for 0 hour, 12 hours, 24 hours, or 24 hours with 500 U/mL recombinant human IFN-γ (R&D Systems, USA) added for the last 12 hours of culture. Cells were lysed, and total RNA was prepared using an RNeasy Mini Kit (Qiagen, USA) according to the manufacturer’s protocol. RNAseq was performed by the Avera Institute for Human Genetics (South Dakota, USA) as follows: total RNA was assessed for degradation on an RNA 6000 Nano chip ran on a 2100 Bioanalyzer (Agilent, USA) where the average RNA integrity score for the sample set was 9.7. Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) following the low sample procedure. Briefly, ribosomal RNA was depleted from total RNA, and the remaining RNA was purified, fragmented appropriately, and primed for cDNA synthesis. Blunt-ended cDNA was generated after first and second strand synthesis. Adenylation of the 3′ blunt-ends was followed by adapter ligation prior to the enrichment of the cDNA fragments. Final library quality control was carried out by evaluating the fragment size on a DNA1000 chip ran on a 2100 BioAnalyzer (Agilent, USA). The average library yielded an insert size of 326 base pairs (bp). The concentration of each library was determined by quantitative PCR by the KAPA Library Quantification Kit for Next Generation Sequencing (KAPA Biosystems, USA) prior to sequencing. Libraries were normalized to 2 nM in 10 mM Tris-Cl, pH8.5 with 0.1% Tween 20 then pooled evenly. The pooled libraries were denatured with 0.05 M NaOH and diluted to 20 pM. Cluster generation of the denatured libraries was performed according to the manufacturer’s specifications (Illumina, USA) using the HiSeq PE Cluster Kit V.2 chemistry and flow cells. Libraries were clustered appropriately with a 1% PhiX spike-in. Sequencing-by-synthesis was performed on a HiSeq2500 using V.2 chemistry with paired-end 101 bp reads resulting in an average of 52.4 million paired-end reads per sample. Sequence read data were processed and converted to FASTQ format for downstream analysis by Illumina BaseSpace analysis software, FASTQ Generation V.1.0.0.

RNA sequencing data analysis

Quality control of cell line RNAseq data was performed using FastQC V.0.11.5, FastQ Screen V.0.11.1, RSeQC V.3.0.0, MultiQC V.1.6 and proprietary algorithms of the BostonGene platform.55 RNAseq reads were pseudoaligned using Kalisto V.0.42.4 to GENCODE V.23 transcripts.56 Transcripts with transcript type in (protein_coding, IG_C_gene, IG_D_gene, IG_J_gene, IG_V_gene, TR_C_gene, TR_V_gene, TR_D_gene, TR_J_gene) were selected, then non-coding RNA transcripts and histones and mitochondrial transcripts were removed resulting in 20,062 protein coding genes. Gene expressions were quantified as transcripts per million and log2 transformed.57 Gene expression changes in cell lines treated with EGFRi were shown as relative (log) expression normalized to untreated control cells. PROGENy V.1.4.1 was used to calculate seven pathways activity scores (EGFR, MAPK, PI3K, TRAIL, TNFa, NFkB, and JAK-STAT).58 Other pathway signature scores were calculated using ssGSEA using in-house python implementation. The pathways activity were represented as gene signatures, downloaded from mSIGdB V.6.2,59 unless other specified: ‘Cell cycle signature’ - HALLMARK_G2M_CHECKPOINT, ‘Apoptosis signature’ - HALLMARK_APOPTOSIS, MYC HALLMARK_MYC_TARGETS_V2, ‘EMT signature’ -HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, ‘IFN-γ signature’ - HALLMARK_INTERFERON_GAMMA_RESPONSE, ‘HLA expression’ - gene set (HLA-A, HLA-B, HLA-C). Pathway score differences relative to the control were normalized to the maximum values in each pathway separately and displayed on the line plots. For schematic representation, the maximum absolute deviation of the pathway activity score change from the control point (0 hour) to the 24 hours time point was calculated within each pathway. Pathway colors on the schema correspond to the percent of the maximum absolute deviation. Heatmaps, dot plots, line plots, and bar plots were created using pandas V.0.23.4, matplotlib V.2.1.1 and seaborn V.0.9.0 python packages.60

Luminex assay

Duplicate samples of supernatants from untreated and 1 µM EGFRi-treated H1975 and H1299 cell lines were analyzed for the presence of CCL2, CXCL1, CXCL2, CXCL8, IP10, IL1RA, IL6, VEGFA, CSF2, and CSF3 proteins using a custom Luminex kit according to the manufacturer’s instructions (R&D Systems, USA). Fifty microliters of test supernatant and the appropriate microparticle cocktails were added (1:10 dilution) to each well and incubated for 2 hours in a microplate shaker at room temperature. Plate was washed three times, and 50 µL of Biotin-Antibody cocktail (1:10 dilution) was added and incubated for 1 hour, followed by three washes. After incubating with 50 µL of Streptavidin (1:25 dilution) for 30 min, the plate was washed three times, and microparticles were resuspended in buffer and the plate was read using a Luminex plate reader. EGFRi-induced changes were expressed as fold increase or fold decrease compared with measured baseline (0 hour) concentrations. In cases where concentrations measured fell below the level of detection, the minimum threshold of detection according to the manufacturer was used.

T cell functional assays

Statistical analysis

All statistical analyses were performed using the GraphPad Prism 5 or 8 (GraphPad Software, La Jolla, California, USA). Survival curves were calculated using Kaplan-Meier estimate, and OS was measured from the date of enrollment up to 31 December 2018 or the time of death. Two-tailed Student’s t-test or Mann-Whitney U test or two-way analysis of variance test with multiple group comparison adjustment (Dunnett’s test) was used to analyze the statistical significance between groups. A p value less than or equal to 0.05 was the threshold used to determine statistical significance.