Patients underwent leukapheresis in order to isolate peripheral blood mononuclear cells (PBMC). PBMCs were incubated in a humidified incubator for 1 hour at 37OC to allow plastic adherence. The adherent cell fraction was used for DC culture by incubation for 5 days in X-VIVO15 Medium supplemented with 1% autologous heat-inactivated plasma, 1,000-U/mL GM-CSF and 250-U/mL IL-4. Maturation of DCs were performed on days 5–7 with 1,000 -U/mL TNF-a, 1,000 U/mL IL-1b, 1,000-U/mL IL-6 and 1-mg/mL PGE2.
Cells were harvested on day 7 and transfected with mRNA. Mature DCs were washed twice, suspended in Opti-MEM medium and adjusted to a final cell density of 6.25×107 cells/mL. The cell suspension (800 mL) was preincubated in a 4-mm gap electroporation cuvette for 5 min on ice. 20 mg of mRNA encoding p53, survivin, or hTERT were transferred to the cuvette and DCs were pulsed using a BTX 830 square-wave electroporator. Electroporation settings were adjusted to a single pulse of 500 V and 2 ms. After electroporation, DCs were rested for 30 min in 37OC X-VIVO 15 before frozen in aliquots of 1×107 DCs in 85% autologous serum, 10% DMSO, and 5% Glucosteril 40% using automated cryopreservation.
Six cycles of cyclophosphamide 50 mg orally bi-daily for a week every second week (day 1–7). During the six cycles patients received at least 5×106 autologous DCs administered by intradermal (i.d.) injection in the week without
The treatment was well tolerated. Three CTC grade III events were reported. Following leukapheresis, one patient had a lung embolus, which was associated with the catheterization. Anticoagulant treatment was applied, and the patient recovered completely. Another patient had a lung embolus and a grade III pleural effusion due to his malignant disease. Apart from these events, only CTC grade I/II events were reported. All the toxicity was manageable.
Eighteen patients were evaluable after six cycles. Of these, nine patients had progressive disease (PD) as best response and nine patients achieved stable disease (SD). In three patient’s minor tumor regression was observed. By IFNg ELISpot and proliferation assay immune responses were seen in 6/17 and 4/17 patients, respectively; however, no correlation with clinical response was found. The percentage of Tregs was unchanged during treatment.
Median PFS for the 22 patients was 3.1 months (95% CI 2.8–3.4) and median OS was 10.4 months (95% CI 1.5–19.3).
In conclusion, treatment with autologous DCs transfected with p53, survivin, and hTERT encoding mRNA and concomitant cyclophosphamide regimen was feasible and well tolerated. Sign of clinical benefit was seen, as 9 of 22 (41%) treated patients achieved SD and 3 of 18 evaluable patients experienced tumor shrinkage.
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Scientific article publishing date : 26/8/2016
Immucura identifier : BSC21_045EN