We included patients aged between 20 and 75 years with histologically or cytologically diagnosed NSCLC, and who had received platinum-based chemotherapy, as well as an appropriate tyrosine kinase inhibitor for those with an epidermal growth factor receptor (EGFR)-sensitizing mutation. Additional inclusion criteria were as follows: a performance status of 0 or 1; the existence of a measurable lesion; expected survival of 3 months or more; and had received no chemotherapy or radiotherapy for at least 4 weeks before enrollment. Vα24+Vβ11+ iNKT cells were detected at a level of more than 10 cells in 1 mL peripheral blood by flow cytometry. Exclusion criteria included the following: a positive response to HIV, hepatitis C virus, or human T cell leukemia virus antibodies; positivity for hepatitis B surface antigen; the presence of active inflammatory disease or active autoimmune disease; a history of hepatitis; pregnancy or lactation; concurrent corticosteroid therapy; or evidence of another active malignant neoplasm. Histological type, staging, and the antitumor effect of the treatment were classified according to the seventh lung cancer tumor node metastases classification and staging system.26

The study was an open label, single-arm, phase II clinical trial, and was undertaken in the Department of General Thoracic Surgery, Chiba University Hospital, Japan, according to the standards of Good Clinical Practice for Trials on Medicinal Products in Japan. The study was conducted in accordance with the Declaration of Helsinki.

A scheme of the study design is shown in figure 1. Written informed consent was obtained from all patients before undergoing a screening evaluation to determine eligibility. Extensive clinical and laboratory assessments were conducted once a week and consisted of a complete physical examination and standard laboratory assessments. Adverse events and changes in laboratory values were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events V.3.0 (CTCAE). Patients were followed up for 2 years or until death.

All procedures were performed according to Good Manufacturing Practice standards. Eligible patients underwent peripheral blood leukapheresis (COBE Spectra, Terumo BCT, Lakewood, CO), and PBMCs were separated by density gradient centrifugation (OptiPrep, Axis-Shield, Oslo, Norway). For the preparation of APCs containing DCs, PBMCs were cultured with GM-CSF and IL-2, as previously described.22 Briefly, PBMCs were washed three times and resuspended in AIM-V (ThermoFisher Scientific, Waltham, MA) with 800 U/mL human GM-CSF (GeneTech Co., Ltd, China) and 100 JRU/mL recombinant human IL-2 (Imunace; Shionogi, Japan). The cultured cells were pulsed with 200 ng/mL αGalCer (KRN7000; REGiMMUNE, Japan) on the day before administration. After 7 or 14 days of culture, the cells were harvested, washed three times, and resuspended in 100 mL 2.5% albumin in saline. The criteria for αGalCer-pulsed APC administration included a negative bacterial culture 48 hours before APC injection, cell viability of >60%, and an endotoxin test 48 hours before APC injection with a result of <0.7 EU/mL. APCs containing DCs (1×10⁹ cells/m²/injection) were administered four times intravenously (figure 1).

The phenotypes of monocytes and APCs were determined with a FACSCanto II (BD Biosciences, Franklin Lakes, NJ) and FlowJo software (Flowjo, LLC, Ashland, OR). Monoclonal antibodies (mAbs) were as follows: fluorescein isothiocyanate (FITC)-labeled anti-HLA-DR, anti-CD14 (BD Biosciences), phycoerythrin (PE)-labeled anti-CD86, anti-CD1d (BD Biosciences), allophycocyanin-labeled anti-CD40 (BD Biosciences), and Pacific blue (PB)-labeled anti-CD11c (Biolegend, San Diego, CA). Isotype-matched control mAbs were used as negative controls.

PBMC samples were obtained from patients before the administration of αGalCer-pulsed APCs, and another eight times during the 11-week study period (figure 1). mAbs to detect and fractionate mononuclear cells in peripheral blood were: FITC-conjugated anti-TCR Vα24 (Beckman Coulter, Brea, CA), anti-CD14 (BD Biosciences), anti-CD45RA (BD Biosciences), PE-conjugated anti-TCR Vβ11 (Beckman Coulter), anti-CD56 (BD Biosciences), anti-CCR7 (Biolegend), allophycocyanin-conjugated anti-CD3 (BD Biosciences), αGalCer-loaded CD1d tetramer (ProImmune), allophycocyanin-Cy7 conjugated anti-CD3 (BD Biosciences), PE-Cy7-conjugated anti-CD8 (BD Biosciences), PB-conjugated anti-CD4 (BD Biosciences), anti-CD16 (BD Biosciences), anti-PD-1 (Biolegend), and anti-HLA-DR(Biolegend). The absolute numbers of these cells were also calculated using automated full blood counts.

PBMCs were washed three times with PBS and then stored in liquid nitrogen until use. The detection of IFN-γ-secreting cells was performed as previously described.20 Briefly, 96-well filtration plates (Millipore, Bedford, MA) were coated with human anti-human IFN-γ (10 mg/mL, Mabtech, Sweden). PBMCs (5×10⁵/ well) were incubated for 16 hours with αGalCer (200 ng/mL) in 10% fetal bovine serum containing RPMI. Phorbol 12-myristate13-acetate (10 mg/mL) plus ionomycin (10 nM) was used as a positive control. After culture, the plates were washed and incubated with biotinylated anti-IFN-γ (1 mg/mL, Mabtech). Spot-forming cells were quantified by microscopy.

The primary endpoint was overall survival (OS; time from the first apheresis until death from any cause). Secondary endpoints were safety, progression-free survival (PFS), response rate (RR; percentage of patients with complete response (CR) or partial response (PR) as per RECIST V.1.1), and disease control rate (DCR; percentage of patients with CR, PR, or stable disease (SD)). OS, PFS, RR, DCR, and safety were analyzed in all patients who received at least one course of study treatment. Protocol deviation cases were excluded from the correlation analyses of immune responses and survival time because it would not be possible to adequately assess these without two courses of treatment. Because the MST of the previous phase I/II trial was 18.6 months, the expected survival time was determined to be 17 months.22 In addition, the threshold time was set to 8 months with reference to 7.8 months, which was the result of the MST of low-dose docetaxel monotherapy for NSCLC patients in Japan,27 the standard therapy for second-line treatment at the start of this study.

All patients underwent an assessment of tumor status by chest CT at baseline and 3 weeks after the fourth αGalCer-pulsed APC administration (11 weeks after study entry).

Continuous variables are summarized as the mean and SD and categorical variables as frequencies and proportions. The Kaplan-Meier method was used to estimate MST and a 95% CI of OS and PFS. The difference between the good responders group and the poor responders group for OS was evaluated using a log-rank test. The linear mixed effect model for repeated measures was used to estimate the mean, SE, and 95% CI of the mean of each measuring point of immune monitoring, which can address all available post-baseline data. Safety was examined using the safety analysis set, which was defined as any patient who had received at least one dose and who had at least one safety assessment at post-baseline. A two-sided p-value of <0.05 was considered statistically significant, and all analyses were performed using SAS V.9.4 for Windows.

A total of 35 patients were enrolled in the study from February 2012 to August 2015. Three patients became protocol deviation cases due to exacerbation of their primary disease within 4 weeks after administration of the therapy, and another treatment was prescribed. The characteristics of 35 patients who were enrolled in the trial are summarized in table 1. The average age of these patients was 58.5 years. Six patients were clinical stage IIIB, 20 were stage IV, and nine were recurrent after surgical treatment. The study included 25 patients with adenocarcinoma, eight patients with squamous cell carcinoma, one patient with large cell carcinoma, and one patient with atypical carcinoid. All patients had received systemic first-line chemotherapy including platinum-based chemotherapy or EGFR tyrosine kinase inhibitor (TKI). Seven cases were positive for EGFR mutations, and five out of seven cases had received gefitinib as a primary treatment.

The phenotype of APCs containing DCs prepared for each administration was analyzed by flow cytometry and is summarized in online supplementary table 1. In all preparations, cultured cells exhibited an immature and mature MoDCs phenotype expressing HLA-DR and CD86. The expression levels of CD11c, CD1d, and CD40 were modest, while CD14 expression was low.

The primary endpoint was OS in the total population. All patients were followed up for 6.4 to 27.2 months to elucidate the prognosis and cause of death. The MST of all 35 cases was 21.9 months (95% CI, 14.8 to 26.0). The 1-year survival rate was 84.4%, and the 2-year survival rate was 40.6% (figure 2).

Antitumor response was evaluated by chest CT findings as described in table 2. There was one case of PR, 14 of SD, 19 of progressive disease, and one that was unevaluable. RR was 2.9% and DCR was 42.9%. The median PFS was 2.8 months (95% CI, 2.6 to 3.7) and the rate of PFS at 1 year was 4.3%.

Adverse events related to the treatment are listed in table 3. Nine out of 35 patients had treatment-related adverse events. All treatment-related adverse events were evaluated as grade 1 or 2. Dysesthesia is associated with hypocalcemia in peripheral blood leukapheresis, and the symptoms disappeared immediately following the administration of calcium gluconate in this study. All grade 3 or 4 adverse events during the treatment are listed in online supplementary table 2. One patient experienced worsening of tumor pain on day 15 (grade 3) and was admitted to the hospital for pain control. This patient had a metastatic tumor in the retroperitoneal space and morphine was prescribed before study enrollment due to back pain. The rapid enlargement of the mass induced worsening of tumor pain. Thus, no clear relationship between the cell therapy and tumor pain was established. No severe side effects were observed in the other patients.

Three patients experienced increased serum amylase levels (grade 3). Among these three patients, one patient showed grade 2 and two patients showed grade 1 serum amylase on study enrollment. No signs of pancreatitis or sialadenitis were detected, and no cases required additional treatment. One patient experienced grade 3 hypertension after the second apheresis, which occurred transiently. All adverse events that occurred during the treatment period are summarized in onlineonline supplementary table 3.

Immune monitoring was performed for 35 patients who received αGalCer-pulsed APC injections. PBMCs were fractionated, and each subset of immune cells was identified using mAbs and flow cytometry analysis. Figure 3 shows the results of the linear mixed models for repeated measurements of each immune cell number in all 35 cases. The absolute number of white blood cells and lymphocytes significantly increased after treatment; the geometric mean of white blood cells changed from 6.02×10⁶ to 6.74×10⁶ count/mL and that of lymphocytes changed from 1.28×10⁶ to 1.45×10⁶ count/mL (all p<0.001) (figure 3A,B). Moreover, the absolute number of Vα24+Vβ11+ iNKT cells significantly decreased after αGalCer-pulsed APC injection; the geometric mean of iNKT cells changed from 1.74×10² to 8.72×10 count/mL (p<0.001) (figure 3C). The number of natural killer (NK) cells significantly increased; the geometric mean of NK cells was increased from 1.25×10⁵ to 1.71×10⁵ count/mL (p<0.001) (figure 3D). While CD8+ T cells and effector memory CD8+ T cells did not increase, effector CD8+ T cells gradually increased from 5.40×10⁴ to 6.67×10⁴ count/mL during the treatment period using all available data for each patient (p<0.01) (figure 3E–G). However, PD-1+CD8+ T cells did not change significantly.

Because our previous clinical study showed that increased IFN-γ-producing cells were associated with prolonged MST after αGalCer-pulsed APC injection,22 we assessed the ability of PBMCs to produce IFN-γ after restimulation with αGalCer in vitro. The cryopreserved PBMCs were thawed and stimulated with αGalCer for 16 hours in vitro, and enzyme-linked immunospot assay assessed the number of IFN-γ-producing cells in the culture. As a result of the linear mixed model, IFN-γ-producing cells rapidly increased before the fourth αGalCer-pulsed APC administration and were back to the equivalent value of the pretreatment state 3 weeks after two cycles of the treatment (figure 4A). In 24 patients, the number of cells producing IFN-γ increased more than 1.9-fold after the administration of αGalCer-pulsed APCs (good responder: solid line in figure 4B). In the remaining eight patients, a minimal alteration (<1.9) in the IFN-γ-producing capacity was observed (poor responder: dotted line in figure 4B). Among 35 cases, good responders exhibited a trend of longer survival (MST, 24.4 months; 95% CI, 15.8–not estimable) compared with poor responders (MST, 14.9 months; 95% CI, 9.6 to 22.9; p=0.0656) (figure 4B). The 2-year survival rate of the good responder group was 50.0% and that of the poor responder group was 12.5%. iNKT cell number before treatment did not correlate with good immune responses or good prognosis (data not shown ).