Trial design and clinical setting
The trial (registered at clinicaltrials.gov: NCT02689644) was designed as a prospective, single-armed, open-label phase 1 study to assess safety and efficacy of ilixadencel injection in subjects with advanced unresectable and/or metastatic progressing GIST despite ongoing treatment with second or later line of TKI treatment. The initial trial (Fig. 1) was designed to consist of two cohorts (modified 3 + 3 design per cohort) and 12 patients in total, with one arm receiving a dose-escalated schedule. Following inclusion of six patients, the trial was closed due to slow subject recruitment. All included patients followed the arm with a non-dose-escalated schedule.
The trial was conducted at the Department of Endocrine and Sarcoma Surgery, Karolinska University Hospital, Stockholm, Sweden. It was approved by the institutional review board (Dnr 2015/1619-31), as well as the Swedish Medical Products Agency (Dnr 5.1-2015/77670). All included patients signed an informed consent, in line with Declaration of Helsinki, prior to study participation. The data safety monitoring board (DSMB) included a minimum of two independent physicians with relevant expertise in oncology and clinical research at all times. Based on safety information, the DSMB directed recommendations to the Sponsor concerning continuation, modification, and trial termination.
Men and women, at least 18 years of age, with a diagnosis of unresectable or metastatic GIST that had progressed despite second-, third-, or fourth-line treatment with a TKI were considered. The size of the lesion had to be of at least 3 cm in longest unidimensional diameter measured by computed tomography (CT). Patients were excluded if performance status according to Eastern Cooperative Oncology Group (ECOG) was > 2, abnormal hematological parameters. Patients with viral disease (hepatitis B, C, and HIV), active autoimmune disease which required immunosuppressive, or with previously major reaction/adverse event (AE) in connection with previously made vaccinations or transfusion of blood products were excluded from study inclusion. For detailed inclusion and exclusion criteria.
Preparation of ilixadencel
Ilixadencel was prepared from healthy blood donors, in which donor screening and donor eligibility are regulated by country-specific law and implemented EU directives. Cells in the leukapheresis and thereafter fractionated by elutriation in a close system, ELUTRA® (Gambro BCT). The elutriation results in a cell product in fraction 5 that contains > 90% CD14+ monocytes. These cells are used for differentiation (using the well-established differentiation cocktail GM-CSF plus IL-4) and activation (R848.poly-IC and IFN-gamma) into proinflammatory DCs. The final drug product is cryopreserved cells formulated in human plasma and 10% DMSO . The requirements of the product post-thawing were a cellular viability of > 70%, HLA-DR expression of > 50%. Furthermore, it was required to produce > 7500 pg RANTES/mL/106 cells.
The ilixadencel batch used for the first four GIST patients was produced at Cancer Center Karolinska, Karolinska University Hospital, Sweden. Immediately before administration to the patient, the frozen vials were thawed and the cells were washed and resuspended in 0.15 M saline with 2% human serum albumin before administration. The last two patients received ilixadencel from a batch produced at BioNTech, Idar-Oberstein, Germany (after a standard tech transfer). These cells were used as a direct-injectable product after thawing without any additional preparation steps prior to the intratumoral administration.
The dosing regimen was chosen from a previous first in-human trial in metastatic renal cell carcinoma patients  where doses of 5 × 106 (low dose), 10 × 106 (medium dose), and 20 × 106 (high dose) were used. A combination of safety, immunological, and efficacy parameters were considered when selecting the medium dose of 10 × 106 cells for this trial.
The first dose of ilixadencel dose containing 10 × 106 viable HLA-DR+ cells was injected on study day 1, and the second dose on day 14 ± 3 days. The starting dose could be reduced to 5 × 106 cells for subjects where limiting toxicities (LTs) were observed. The injections were ultrasound-guided; all injections were done at the Department of Radiology at Karolinska University Hospital by experienced radiologists. The target lesions were decided at the discretion of the radiologist. Practically, the injected lesions were also evaluated for size, localization, and viable tumor tissue (i.e., intratumoral vasculature). After injection, patients were observed for at least 6 h for possible adverse events following injection of ilixadencel. The patients continued TKI treatment after ilixadencel administration. If progression occurred until the 3 month screening visit, the subject performed an End of Study visit. If stable disease, the subject continued with TKI and follow-up until progression of disease, and controls as outlined in the protocol.
Primary and secondary objectives
The primary objectives were to evaluate ilixadencel’s safety profile and identify LTs, if any. Secondary objectives were to evaluate tumor response by CT, evaluate progression-free survival, changes in ECOG score, and to evaluate potential auto- and alloimmunization. Blood samples for immuno-monitor analysis were obtained at screening, at baseline and at 3-month post-vaccination visit.
Safety and toxicity
Adverse events (AEs) were monitored throughout the study and graded according to the National Cancer Institute (NCI) common toxicity criteria (CTCAE) version 4.03. At each trial visit in the clinic, vital signs, physical examination, as well as safety lab were collected. Before each injection, additional blood samples were drawn for hemoglobin, white blood cell count, platelets, and coagulation status. Vital signs were taken at all trial visits. After injection, more thorough monitoring was employed for 6 h.
To evaluate potential treatment-induced alloimmunization at the humoral level, and possible autoimmunization, blood samples were collected at baseline and 3-month clinical follow-up. The detection of donor (vaccine cell)-specific alloantibodies was analyzed with cytometry-based (Luminex) technique. Serum samples for this assay were collected twice, before the first vaccination and at the 3-month follow-up visit. If alloantibodies specific for MHC class I or class II antigens on the vaccine cells were not present before vaccination but were present in the 3-month follow-up, the results were considered as vaccine-induced. For autoimmunization, screening of the following nuclear antigens was performed: SSA (Ro52 and Ro60), SSB, Sm, RNP68, Scl-70, centromeres, and Jo-1 in serum.
Evaluation of tumor response
Tumor response was evaluated by CT (also MRI was acceptable modality, if MRI was chosen, it was the preferred modality for follow-up scans) at baseline and thereafter at 3-month intervals, if no progression occurred, after the first dose of ilixadencel until 12 months after first vaccination. The baseline imaging was undertaken within 28 days before the first injection, and was considered as the baseline measure in the trial. The tumor stage was classified as progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR) according to modified response evaluation criteria in solid tumors RECIST 1.1 and Choi criteria. All CT-evaluated lesions consisted of one injected lesion and one non-injected lesion. In MRI evaluation (one patient), three lesions were used, one injected lesion and two non-injected lesions.
All endpoints are evaluated by descriptive methods.