A standard operating procedure for the identification of preclinical tests for cellular vaccines and release criteria for DC vaccines has been generated in our lab.

Draft templates for IMP have been generated.

Draft templates for CTP have been generated.

We possess an SOP for the assessment of Th subsets via flow cytometry.

(Basic manufacturing and QC SOPs (Generation of Peptide loaded DCs, Generation of cytokines and media components, Generation of autologous plasma and serum, Staining of cells for FACS Analysis, Cell Counting by Trypanblue Staining) have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )

We have worked out SOPs for the cytometric assessment of Th-cell immunity based on antigen-induced CD154 expression, a technology developed by us in the frame of previous DC-THERA activities. Now the entire antigen-specific Th-cells specific for defined antigens can be monitored simultaneously with respect to quantity and quality. Tests can either be performed on standard 4 colour cytometers or on 12 colour high-end cytometric analysers. Tests that can be either performed to analyse fixed or isolate live antigen-specific Th-cells have been adapted also to be used ...

A retrotranscriptional reaction is performed on RNA extracted from T cells or DCs. Transcripts for interested genes are quantified by real-time quantitative PCR on an Perkin-Elmer Applied Biosystems with predesigned TaqMan Gene Expression Assays according to the manufacturer’s instructions.

Untouched naive CD4+ T cells are isolated from CD14- PBMC with a combination of magnetic sorting.

(Basic manufacturing and QC SOPs on the Generation of Peptide loaded DCs have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )

(Basic manufacturing and QC SOPs on the Generation of cytokines and media components have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )

We submitted descriptions of protocols (SOPs exist) used for expanding patient derived T cells into tumor reactive CTLs, based on our special skills in monocyte isolation by elutriation or adherence methods and in DC-T cell co-cultures as well as in immunomonitoring (all applied in our clinical studies described in the knowledge portal). We also included information on transfection of mature DC by electorporation or using adenoviral constructs.

Cytokine production by DCs and T cells is assessed by ELISA assay according to the manufacturer’s instructions of ELISA kit. After the times indicated, supernatants were collected and cytokine detection was performed. MesoScale Assay 7-spot (Meso Scale Discovery) or Luminex® Assay (Invitrogen) were used for detection of IL-1beta, IL-8, IL-6, TNFalpha, IL-10, IL-12p70, IFNg and IL-17A according to the manufacturer’s instructions. These procedures allowed simultaneously measurement of different cytokines in the same supernatants. The multiplex assay format di...

(Basic manufacturing and QC SOPs on the Generation of autologous plasma and serum have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )

We have a clinical protocol for immunotherapy of melanoma patients in preparation, however many details are not yet finalized. Everything is carried out under GMP conditions according to SOPs. In brief, patients with stage III or stage IV melanoma will undergo surgical resection of a melanotic lesion and leukapheresis will be performed on the following day. DCs will be generated from monocytes after elutriation of the leukapheresis product. On day 5 of culture immature DC will be pulsed with irradiated tumor cells (apoptotic bodies) prepared from the removed le...

(Basic manufacturing and QC SOPs (Generation of Peptide loaded DCs, Generation of cytokines and media components, Generation of autologous plasma and serum, Staining of cells for FACS Analysis, Cell Counting by Trypanblue Staining) have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )