Audencel Immunotherapy Based on Dendritic Cells Has No Effect on Overall and Progression-Free Survival in Newly Diagnosed Glioblastoma: A Phase II Randomized Trial

Dendritic cells (DCs) are antigen-presenting cells that are capable of priming anti-tumor immune responses, thus serving as attractive tools to generate tumor vaccines. In this multicentric randomized open-label phase II study, we investigated the efficacy of vaccination with tumor lysate-charged autologous DCs (Audencel) in newly diagnosed glioblastoma multiforme (GBM). Patients aged 18 to 70 years with histologically proven primary GBM and resection of at least 70% were randomized 1:1 to standard of care (SOC) or SOC plus vaccination (weekly intranodal application in weeks seven to 10, followed by monthly intervals). The primary endpoint was progression-free survival at 12 months. Secondary endpoints were overall survival, safety, and toxicity. Seventy-six adult patients were analyzed in this study. Vaccinations were given for seven (3–20) months on average. No severe toxicity was attributable to vaccination. Seven patients showed flu-like symptoms, and six patients developed local skin reactions. Progression-free survival at 12 months did not differ significantly between the control and vaccine groups (28.4% versus 24.5%, p = 0.9975). Median overall survival was similar with 18.3 months (vaccine: 564 days, 95% CI: 436–671 versus control: 568 days, 95% CI: 349–680; p = 0.89, harzard ratio (HR) 0.99). Hence, in this trial, the clinical outcomes of patients with primary GBM could not be improved by the addition of Audencel to SOC.

. Analysis of potential influence of number of vaccinations received on overall survival (OS). In a Pearson correlation calculation, a non-significant trend towards better OS based on the number of vaccinations received can be registered (p = 0.081).  Settings and locations where the data were collected 11 Interventions 5 The interventions for each group with sufficient details to allow replication, including how and when they were actually administered

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Outcomes 6a Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed 11-13 6b Any changes to trial outcomes after the trial commenced, with reasons -Sample size 7a How sample size was determined 13 7b When applicable, explanation of any interim analyses and stopping guidelines -Randomisation: Sequence generation 8a Method used to generate the random allocation sequence 11 8b Type of randomisation; details of any restriction (such as blocking and block size) 11 Allocation concealment mechanism 9 Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned 11 Implementation 10 Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions

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Blinding 11a If done, who was blinded after assignment to interventions (for example, participants, care providers, those assessing outcomes) and how If relevant, description of the similarity of interventions -Statistical methods 12a Statistical methods used to compare groups for primary and secondary outcomes 13 12b Methods for additional analyses, such as subgroup analyses and adjusted analyses 13

Results
Participant flow (a diagram is strongly recommended) 13a For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome Sources of funding and other support (such as supply of drugs), role of funders 13 * We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration for important clarifications on all the items. If relevant, we also recommend reading CONSORT extensions for cluster randomised trials, non-inferiority and equivalence trials, non-pharmacological treatments, herbal interventions, and pragmatic trials.
Additional extensions are forthcoming: for those and for up to date references relevant to this checklist, see www.consort-statement.org.

Immunotherapy: Description of the Audencel DC vaccine
Audencel is a DC-based autologous cancer vaccine. It is comprised of DCs charged with tumorderived antigens that are matured via LPS and IFNγ and that are characterized by the secretion of IL-12. After production, the final DC vaccine product is 1-5 x 10 6 autologous, "semi-mature" DCs in a DMSO-containing freezing medium (CryoStore CS2/Lite, STEMCELL Technologies, Vancouver, CA).

Immunotherapy: Prior studies on Audencel
The DC vaccine technology behind Audencel has been investigated in prior preclinical studies and in a phase I clinical trial [1][2][3][4]. Preclinically, Felzmann et al. showed in human in vitro experiments that maturation of DCs with LPS and IFNγ leads to an immunostimulatory phenotype (characterized by IL-12 secrection) that can efficiently triggered cytolytic activity in autologous T lymphocytes. Importantly, this was only the case for co-cultures performed 2-6 h after maturation stimulus (LPS/ IFNγ) but not for co-cultures performed at 48 hours [1]. The Audencel technology thus uses DCs matured for 6 hours, called "semi-mature". Hüttner et al. studied the DC generation technique used for Audencel in a syngeneic murine in vivo model and found that the IL-12 secreting DCs used could reduce tumor growth (murine cell line K-Balb) [2]. The method for generating DCs from peripheral blood monocytes was evaluated in further human in vitro experiments by Felzmann et al [3]. Finally, the Audencel technology was also tested in a phase I clinical trial where pediatric cancer patients suffering from advanced solid pediatric malignancies were vaccinated by Dohnal et al [4]. This study established feasibility and safety. As a continuation of these early preclinical and clinical experiences, the here presented phase II trial of Audencel applied to patients suffering from glioblastoma was initiated.

Immunotherapy: Production of the Audencel DC vaccine for the phase II clinical trial on glioblastoma
Tumor samples were harvested through surgical resection, irradiated with 12,000 rad (in accordance with local guidelines for the irradiation of blood products for human transfusion) and then stored subsequently without further delay at 4°C and transported to our Good Manufacturing Practice (GMP) facilities under sterile conditions for the generation of autologous tumor lysate. For that, tumor tissue was kept in Phosphate Buffered Saline (PBS; Hyclone, ThermoScientific, Utah, USA), was disrupted mechanically via a scalpel, pressed through a nylon mesh and the resulting cells in single cell suspension were lysed by five freeze/thaw cycles (liquid nitrogen, −150°C) in distilled water resulting in tumor cell lysate ready for further use. Particulate components were removed by centrifugation. Protein concentration of each tumor lysate was determined by Bradford assay and the vials containing protein lysate were kept frozen at −80°C.
Peripheral blood mononuclear cells (PBMCs) were obtained by leukocyte apheresis (performed at the Transfusion Medicine departments of the respective treatment centers according to local protocols and yielding 4-10 × 10 9 mononuclear cells) followed by elutration (Elutra cell separator, Gambro BCT, Inc. Lakewood, Colorado, USA) for the selective enrichment of clinical-scale monocytes. Then, monocytes were cultured in vitro in Cellgro medium (CellGenix Technology, Freiburg, Germany) with the presence of (317U/ml) recombinant human interleukin-4 (IL-4, CellGenix Technology, Freiburg, Germany) and (1000U/ml) recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF, CellGenix Technology, Freiburg, Germany) at a density of 1x10 6 monocytes/cm 2 . On day 3, fresh medium containing the same cytokines, at the same concentration was added. On day 6, immature dendritic cells were incubated with autologous tumor lysate (see above) together with the immunological adjuvant Keyhole Limpet Hemocyanin (KLH, Calbiochem, Darmstadt, Germany) for 2 hours prior maturation stimulus. Subsequently, the DCs were incubated with LPS (200U/ml, E. coli strain O111:B4, Calbiochem, San Diego, CA, USA) and IFNy (50ng/mL, Boehringer Ingelheim,Vienna, Austria) for 6 hours to induce functional maturation.

Immunotherapy: Quality control
At the arrival of tumor material for vaccine production at the GMP facility, a sterility test was immediately conducted (BACTEC system, BD Biosciences, NJ, USA). Only if sterility could be proven, the material was processed further. Two aliquots of each final vaccine batch after production (see above) were again used for quality control that included tests for viruses, mycoplasma, and bacteria according to standard clinical guidelines. In addition, functional potency and the phenotype of the tumour lysate-loaded DCs was examined in vitro.
The To ensure functional potency prior to application to patients, IL-12 production capacity and Tcell stimulation capacity were determined for each batch of the DC vaccine. For IL-12 measurement, an ELISA test system was used. Briefly, one vial of the vaccine was thawed, and the cells were used 24 hours after thawing. 96-well plates were coated with capture antibody (BD, San Jose, CA) diluted in PBS with 0,02% sodium azide. The next day, unspecific binding in the wells was blocked with 2% BSA in PBS. After blocking, IL-12 standard solutions (BD, San Jose, CA) of known concentration (30-1250 pg/ml) and diluted samples (1:2; 1:20; 1:50) were distributed into the wells. On the third day, captured cytokines were detected by primary incubation with a biotinylated detection antibody (BD, San Jose, CA) and secondary via incubation with alkaline phosphatase-conjugated streptavidin (Chemicon, Temecula, C). When phosphatase substrate at a concentration of 1 mg/ml in diethanolamine buffer was added, the respective yellow colour reaction developed. The diethanolamine buffer consisted of 1 M diethanolamine and 0,5 mM MgCl2 diluted in sterile water with a pH of 9.8. The optical density was measured with an ELISA reader (Anthos, Salzburg, Austria) at a wavelength of 405 nm and a reference wavelength of 690 nm. The cytokine concentrations was calculated using the WinRead V.2.3 software. The release criterion for application of the batch for human use in the trial was >100pg/ml IL-12.
For measurement of T-cell stimulation capacity, allogeneic mixed leukocyte reactions (alloMLR) were carried out. Briefly, allogeneic responder peripheral blood mononuclear cells (PBMCs) collected from healthy donors were isolated by gradient centrifugation from peripheral blood and recovered in AIM-V medium (ThermoFisher, Waltham, MA) supplemented with 2% human plasma (Octapharm, Vienna, Austria). Stimulating DCs (10.000, 2.000, or 400) were placed in triplicates (100 μl per well) on a 96 well round-bottom plate and 10 5 responder cells in 100 μl medium were added to each well. For a positive reference 10 5 responder cells were stimulated in 100 μl medium with Staphylococcal enterotoxin A/B (SEA/SEB, Toxin Technologies Inc., Sarasota, FL) at 100 ng/ml final concentration. On day 4 of the co-culture, 1 μCi of tritium thymidine solution (NEN Life Science Products, Boston, MA) was added to each well and the cells were incubated for another 18 hours. Finally, the cells were harvested with a Skatron harvesting device (Skatron, Lier, Norway) and the incorporated tritium thymidine was counted on a Trilux β-plate reader (Wallac Oy, Turku, Finland). The release criterion for application of the batch for human use in the trial was a T-cell proliferation of at least 30% of the reference SEA/SEB response (for the DC:T-cell ratios 1:5 and 1:10 and at least 15% for the DC:T-cell ratio 1:20).
Summing up, all batches of the DC vaccine that were released to the patient had shown IL-12 production capacity and T-cell stimulation capacity as well as a pre-defined stimulatory phenotype and absence of contamination with pathogens. After quality control, the personalized, autologous DC vaccine for each patient was then kept frozen until application. At the time of treatment an aliquot of the DC cancer vaccine containing approximately 1-5 million DCs was thawed and inoculated to the corresponding patient by ultrasound-guided injection intranodally into a tumor-free (cervical) lymph node.

Immunotherapy: Treatment schedule
All patients received the first line standard therapy for GBM: surgery, radiotherapy, and chemotherapy (Temozolomide). Randomization was done following surgery; patients in the treatment arm who received Audencel as an add-on to the standard treatment underwent leukocyte apheresis within 7-14 days after surgery. The first 4 immunizations were administered in weeks 7-10. Six more immunizations were applied in between the 6 blocks of maintenance chemotherapy. After completion of that schedule, patients received boost immunizations every 3 months. The vaccine was applied intranodally; each vaccine aliquot of Audencel contained 1-5 x 10 6 DC. The immunization schedule continued unaltered even if patients suffered disease recurrence and Temozolomide was withdrawn and replaced with an alternative therapy such as Bevacizumab. Patients of both groups received supportive care for acute or chronic toxicity whenever indicated.