Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma

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 [25].

FFPE tumor samples were evaluated for MGMT methylation by MDxHealth (Liege, Belgium) using RT-PCR as previously described [26] 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.

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.

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 CD8^hi and CD8^lo subpopulations, as recommended by Cancer Immunotherapy Consortium Immune Monitoring Panels[27], 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 CD8^hi 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 [2], 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.

Twenty-one patients who were HLA-A1 and/or HLA-A2 positive, 16 males and 5 females, were enrolled between May 2007 and January 2010 (Table 1). There were 17 ND-GBM patients (16 evaluable; one patient did not receive treatment), three patients with recurrent GBM, and one with brainstem glioma (Table 1). A total of 62 dendritic cell vaccinations were administered. In general, the vaccinations were well tolerated with only grade 1 or 2 adverse events (Table 2). The median age was 52 years (range 26–79 years), and the median Karnofsky score was 90 (range 70–100). ND-GBM patients that subsequently progressed were treated with a variety of additional therapies including bevacizumab, CPT-11, dose-dense temozolomide and cediranib.

Pathological examination of tumors showed variable morphologic features of glioblastoma multiforme (GBM). MGMT methylation by PCR and PTEN expression by immunohistochemistry were performed on the majority of cases. (Table 3). MGMT methylation was observed in 60 % (6 of 10) of the newly diagnosed patients and in 47 % (7 of 15) of all the patients with evaluable results. Six of the newly diagnosed patients did not have samples available for testing or had invalid results. PTEN expression by immunohistochemistry was retained in 8 of 15 (53 %) newly diagnosed patients evaluated.

Expression of tumor-associated antigens using PCR measurement of mRNA was assessed on fresh-frozen samples from patient tumors. Previous studies have demonstrated the protein expression of these antigens on primary tumors and GBM cell lines [19‐23]. All patient tumors expressed at least three of the antigens (Table 3). Fourteen (74 %) tumors expressed all six antigens, two (11 %) expressed five antigens, two (11 %) expressed four antigens, and one tumor (5 %) expressed three antigens.

Quantitative expression of the TAA targeted by the vaccine was analyzed in 13 ND-GBM patients for whom these data were available. In this analysis, expression of AIM-2 and MAGE1 was shown to be significantly correlated with PFS and OS (Fig. 2) and expression of HER2 and gp100 showed a trend toward longer PFS and OS. Significant correlations were not observed with IL13Rα2 and TRP-2. FFPE samples from second surgeries in five patients were evaluated and compared with samples from primary tumors. Studies comparing matching FFPE and fresh-frozen samples with PCR measurement of antigen expression (not shown) showed a lower sensitivity of detection using FFPE samples resulting in samples that were negative for expression. Figure 3 shows the expression of target antigens in primary and recurrent tumors from four HLA-A2 patients. Significant post-vaccine downregulation (p = 0.023 by Fisher’s exact test) of the A2 epitopes relative to upregulation was observed in three patients where responses were positive. For comparison, downregulation of the HLA-A1 target antigen AIM2 was not significant in these patient tumors.

CD133, a non-targeted antigen expressed on the stem cell fraction in gliomas and reported to be increased in recurrent tumors [14, 16], was evaluated in FFPE samples from primary and recurrent tumors. In recurrent patients, CD133 expression in the recurrent tumor decreased by two logs in one patient (#03) and became negative in the other (#08) (Fig. 4). In newly diagnosed patients, one patient (#10) was negative for CD133 in both the primary and recurrent tumor, one patient (#09) showed a one-log decrease in CD133 expression, and the third patient (#19) was negative for CD133 in the second surgical sample. This surgery performed on patient #19 showed no evidence of tumor, and this patient was considered not to have progressed.

For survival analysis, only the 16 newly diagnosed adult GBM patients who received treatment were considered. No patient was lost to follow-up. A summary of demographics, survival, immune response, and KPS on individual newly diagnosed patients is presented in Table 4. The progression-free survival (PFS) and overall survival rates (OS) are presented in Table 5. The PFS time was 16.9 months, and a two-year PFS rate was 43.8 % (95 %CI, 19.8–66.0). The three-year overall survival rate was 55.6 % (95 %CI, 28.6–75.9). The median OS was 38.4 months with eight of 16 patients alive. Six patients showed no tumor recurrence at 49 to 66 months. The Kaplan–Meier probability curves of PFS and OS are shown in Fig. 5a and b, respectively. No difference in survival was observed between HLA-A2+ and HLA-A1+ patients.

IFNγ and TNFα production was quantified according to “Patients and methods” (Day −7 and Day 56, respectively). TNFα responses were highly correlated with IFNγ responses (r = 0.92; p = 0.001). Individual patient immune response indexes are presented in Table 4 with representative gating and responder IFNγ plots presented in Fig. 6. For vaccine response determination, post-vaccine values were divided by pre-vaccine values, with a ≥.5-fold increase over pre-vaccine constituting post-vaccine response enhancement. The baseline for the pre-vaccine calculation is cytokine production in the absence of peptide with ≥.5-fold increased production before vaccination constituting and endogenous antigen-directed response as previously published [2, 6]. Antigen-specific cytokine production was attempted by co-staining with immunizing epitope-specific pHLA multimers (NCI), but failed to exhibit convincing pHLA staining in any patient (not shown). Although Day 56 was previously reported to represent the peak response time in similar DC trials [2], immune responsiveness was assessed at later time points in patients with long survival at Day 56. Of these six patients, only one exhibited a previously undetected vaccine response approaching a 1.5-fold increase after Day 56 (1.46-fold; not shown). Analysis of immune responses from samples 7 days before vaccine administration and compared with samples 56 days after the first vaccine showed 5 of 15 GBM patients tested (33 %) exhibited a positive vaccine response of >.5-fold increase. PFS exhibited a trend toward better survival in responders relative to non-responders, as well as increased long-term and overall survivors, but these did not reach significance.