The buffy coat cells of patients were collected by leukapheresis (in 14 out of 21 times DC preparation) or bag method (in seven times). Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density centrifugation. Monocytes were isolated by culturing PBMNCs in plastic culture dish at a cell concentration of 3–5×106/ml and removing non-adherent cells from the dish. Immature moDCs were induced from monocytes by culturing plastic adherent cells in the same culture dish containing RPMI-1640 with 5% autologous serum, 100 ng/ml GM- CSF and 10 ng/ml IL-4 for 7 days. Immature moDCs were matured by adding 10 ng/ml TNF-α on day 6. Mature moDCs were collected from culture dish by pipetting and occasionally using cell scraper.
Tumor antigen peptide used for the study was SART1 (EYRGFTQDF, HLA-A*24:02 restricted, GMP grade). SART1 peptide was added to the moDC culture at a concentration of 50 μg/ml during the last 24 hours. In the last three patients, keyhole limpet hemocyanin (KLH: carrier protein for peptide antigen) was pulsed at a concentration of 50 μg/ml together with SART1 peptide for the last 24 hours of moDC culture. On occasion, PBMNCs and peptide-pulsed moDCs were cryopreserved for later in vitro study.
On the day of vaccination, peptide-pulsed moDCs were washed and re-suspended in 500 μl saline with 5% autologous serum and transferred to a 1 ml syringe for injection. The moDC suspension was injected intravenously (IV) in the first four patients (patients 1–4) and subcutaneously (SC) in the upper arm in the last three patients (patients 5–7). Infusions with peptide-pulsed moDCs were repeated every three weeks up to five times depending on the patient.
The vaccination was generally well-tolerated and no allergic reaction to the vaccine was observed. One patient who received SART1 peptide-pulsed moDCs showed a moderate hypophosphatemia, although the relationship with moDC vaccination was not definite.
DTH and effects of DC therapy
Although skin DTH reactions against KLH were detected in two out of three patients vaccinated with moDCs pulsed with SART1 and KLH, DTH reaction against SART1 peptide was not observed in all the seven patients. One patient who received SART1 peptide-pulsed moDC vaccine (patient no. 2) remained stable for 20 months after moDC therapy judging from tumor marker and CT findings and he was categorized as no change (NC). But thereafter he developed lung metastasis, for which the operation was undertaken. The remaining six patients had progressive disease (PD) with the median survival of 3.7 months and no favorable response was observed during and after the vaccination course.
This present study demonstrated that moDCs prepared from advanced stage of squamous cell carcinoma of esophagus possess a good immune function and in vivo immune responses (detected by ELISPOT assay) were evoked by the infusion of these moDCs. These findings suggest that DC‐based immunotherapy could be one of the modalities applicable for squamous cell carcinoma of esophagus.
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Scientific article publishing date : 11/20/2014
Immucura identifier BSC21_181EN