This was an open-label, single-institution, single-arm, phase I study with the primary endpoint to evaluate the immunogenicity of ICT-107 and secondary endpoints of safety and efficacy in patients with glioblastoma or brainstem glioma. ND-GBM patients with surgery, who had no imaging of tumor progression after a standard treatment with concurrent temozolomide (TMZ) and radiation therapy, were eligible for vaccine therapy, and recurrent GBM patients with a gross total resection were also eligible. After explanation of the protocol, written informed consent was obtained from patients before screening; the vaccine was then prepared and administered intradermally in the axilla region every 2 weeks for three consecutive doses after the completion of radiation or surgery. A diagram of the treatment schedule is shown in Fig. 1. Post-vaccine, newly diagnosed patients received maintenance temozolomide chemotherapy following the last dose of vaccine and recurrent patients received chemotherapy with or without bevacizumab. Patients were monitored once a month by neurological examination and every 2 months by magnetic resonance imaging. Progressive disease was defined by MacDonald criteria. This protocol was allowed by the US FDA and the local institution review board at Cedars-Sinai Medical Center.
Patients with newly diagnosed or recurrent GBM with a gross total resection (>95 %) or brainstem glioma were HLA-A1 or HLA-A2 positive (HLA typing done by low resolution PCR-SSOP). Inclusion criteria included the presence of at least one of the vaccine antigens on the patient’s tumor, a Karnofsky score of at least 60 %, glucocorticoid therapy with dexamethasone ≤4 mg/day, and normal baseline hematological parameters. Exclusion criteria included pregnancy; severe pulmonary, cardiac, or other systemic disease associated with an unacceptable anesthetic or operative risk; presence of an acute infection requiring active treatment; history of an autoimmune disorder or allergy to gentamicin; or prior history of other malignancies, excluding basal cell carcinoma and benign tumors. The extent of surgical resection was defined as either complete resection, with no linear gadolinium enhancement on T1 MRI image or sub-total resection in which minimal residual nodules were observed.
Tumor samples collected from surgery were dissociated into single-cell suspensions with trypsin–EDTA (Invitrogen, Carlsbad, CA) for 30 min and frozen at −80 °C. In some samples, formalin-fixed paraffin-embedded (FFPE) samples were evaluated. RT-PCR method and primers used to quantitate expression of mRNA for MAGE1, gp100, HER2, AIM-2, TRP-2, and CD133 were previously described [14, 17, 21–23]. IL13Ra2 primers were from Qiagen, Valencia, CA. Data analysis was done by comparison with the GAPDH control using the 2−ΔΔ CT method .
FFPE tumor samples were evaluated for MGMT methylation by MDxHealth (Liege, Belgium) using RT-PCR as previously described  and PTEN immunohistochemistry with PTEN antibody (Clone 6H2.1, Cascade Biosciences). Immunoreactivity was detected and visualized with either an Ultraview DAB or a Bond Refine DAB system and reviewed by at least one board-certified neuropathologist.
Preparation of autologous vaccine
Monocytes were prepared from leukapheresis products by adherence for 2 h at 37 °C, and adherent cells were cultured for 5 days in RPMI 1640 with 10 % autologous serum supplemented with recombinant human granulocyte–macrophage colony-stimulating factor (Berlex) (800 units/ml) and interleukin-4 (R&D systems) (500 units/ml). On Day 5, 50 ng/ml tumor necrosis factor-α (R&D systems) was added for 3–4 days.
Peptides included HLA-A1 restricted, MAGE1(161) EADPTGHSY, AIM-2(14) RSDSGQQARY, and HLA-A2 restricted, TRP-2(180) SVYDFFVWL, gp100(210M) IMDQVPFSV, HER2(773) VMAGVGSPYV and IL13Rα2(345) WLPFGFILI (Clinalfa, Läufelfingen, Switzerland). Cells from Day 8–9 cultures were cultured at 106/ml with peptides (10 μg/ml per antigen) at 37°/5 % CO2, 16–20 h. Pulsed cells were resuspended at 1 × 107 cells/ml in 10 %DMSO/90 % autologous serum and 1.1 ml, dispensed into 1.8 ml Nunc cryo tubes (Roskilde, Denmark), frozen at 1 °C/min, and stored in the vapor phase of liquid nitrogen.
On culture Day 2, a media aliquot was tested for sterility. A gram stain, sterile culture, mycoplasma, and LAL endotoxin assay were performed on the final product before administration.
The frozen dose was thawed rapidly in a 37 °C water bath, transferred into a 1 ml tuberculin syringe, and administered intradermally at multiple sites in the axillary region. Patients were monitored for 2 h post-immunization for any adverse effects. Patients received pretreatment with 50 mg of diphenhydramine and 650 mg of acetaminophen as needed.
Immune response methods
Pre- and post-vaccination (7 days prior to vaccination, and 56 days later: 5 weeks after administration of the third and last vaccine), PBMCs were thawed and co-cultured with a mixture of all immunizing peptides (10 μg/ml) in stimulating medium (10 % FBS/RPMI+ anti-CD28/CD49d) at 37° C for 7 days.
On Day 7, cultures were restimulated with peptides. Brefeldin-A and monensin were added after 1 h, and the cells were cultured five more hours. Washed cells were stained with surface markers (CD8, clone OKT8; CD3, clone OKT3; CD107a, clone eBioH4A3, IFNγ, clone 4S.B3; TNFα, MAb11; all eBioscience), resuspended in 1X PermiFlow (Invirion) and stored at room temperature overnight. Intracellular staining for IFNγ and TNFα was performed and the CD8+INFγ+ population quantified within both CD8hi and CD8lo subpopulations, as recommended by Cancer Immunotherapy Consortium Immune Monitoring Panels, using identical forward and side scatter gating in all samples (Fig. 6). A proportion of vaccine-enhanced cytokine+ CD8+ cells [(Ag stimulated/unstimulated CD8+ post-vaccine) ÷ (Ag stimulated/unstimulated CD8+ pre-vaccine)] were used to assess immune responsiveness. CD107a staining closely paralleled that of IFNγ and TNFα (not shown) and correlated significantly with IFNγ responses (r = 0.65 for all cytokine+, and 0.932 for CD8hi gates; p = 0.01 and 0.001, respectively). Patients with a 1.5-fold higher post-vaccine than with pre-vaccine score were considered responders. This protocol reproduced immune response results of previous autologous DC stimulation assays analyzed by quantitative PCR for IFNγ production at Day 56 , without requiring high volume blood collections for DC differentiation (not shown).
Continuous variables were compared with Student’s t test and categorical variables compared using Fisher’s exact test. Probability of survival was determined with SAS software by the Kaplan–Meier method, using two-tailed Mann–Whitney log-rank test exclusively to compare groups. Pearson’s correlation coefficients (r values) were calculated in Excel software. Standard errors (±) of the mean were provided whenever appropriate. Significant observations were considered for p < 0.05.