Argos successfully developed a manual DC manufacturing method with excellent reproducibility in terms of quality, yield, and viability of DCs. The process developed has the following unique advantages: - The manufacturing process has been optimized for producing DC from non-frozen day-old pheresis material which allows centralized manufacturing. The pheresis product is sent in a specially designed shipper to the manufacturing facility. - The final vaccine product is formulated for direct injection (i.e., no clinical site participation required except for bedside thaw and i.d. injection)

In 2008, much effort was spent to optimize protocols for RNA transfection in human sDCs. - fusion proteins of the DC specific receptor DC-SIGN fused to GFP - use of rna to express melanoma associated antigens in dendritic cells to prepare better vacines for the treatment of melanoma patients.

A single step procedure for the in vitro maturation and antigen loading of human moDC was optimized. DC were electroporated with mRNA encoding tumor antigens and a putative TLR3 ligand. In vitro stimulation of naïve T cells indicated the superiority of this approach compared to in vitro stimulation with DC matured with inflammatory cytokines followed by electroporation with TAA encoding mRNA.

In ongoing clinical trials (multipeptide loaded cytokine matured moDC +/- CD40L activation in >60 pat.) we have obtained valuable information with high impact on future DC-trials: 1) the presence of 2% DMSO in the thawed final vaccine has no negative impact on the quantity and quality of induced immune responses; thus thawed DMSO containing vials do not have to be centrifuged, but simply diluted; 2) class I peptide loaded cytokine matured DC induce de novo or expand preexisting CD8+ T cell IFN gamma responses in the majority of melanoma patients (>70%), surpri...

We performed experiments to set up the optimal conditions for both transient transfection of synthetic siRNAs and lentiviral transduction of shRNAs into DC. To optimize transfection efficiency, while minimizing toxicity, non targeting FITC-conjugated siRNA were transfected in two different experimental settings: a) fresh monocytes and b) immature DCs (iDCs) obtained from monocytes in the presence of GM-CSF/IL-4. In both experimental settings a considerably high transfection efficiency (up to 75%) was achieved in the absence of significant cell death. Moreov...

In ongoing trials with peptide loaded DC (> 60 pat.) routine immunomonitoring at frequent intervals showed a high correlation between different assays (IFN gamma ELISPOT; modified MLPC = combination of limiting dilution, multiple peptide restimulation, tetramer detection). Both assays demonstrated declining responses upon extension of vaccination intervals. We suggest: IFN gamma ELISPOT as basic assay (costs, workload) that can be frequently performed in every patient for routine monitoring Modified MLPC (combination of limiting dilution, multiple peptide resti...

To determine ROS production we used the modified version by Choi et al. (2006) of the microscopic Nitroblue tetrazolium (NBT, Sigma-Aldrich) assay. Briefly, DCs were stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT, which allows the precipitation of formazan particles in presence of ROS. Blue formazan particles were dissolved using 2M KOH and DMSO and its absorbance was measured using a microplate reader at 620 nm. The absorbance of dissolved NBT increased in proportion to cell number, incubation time, and stimulus concentration.

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 co-coltured with fungi -pulsed DCs and then the polarization is assessed by flow cytofluorimetry and cytokine production.

The vaccine is composed by autologous DCs pulsed with apoptotic autologous ovarian carcinoma cells. Apoptosis is induced by UV.

Protocols were developed to refold in vitro CD1 molecules, which were used for the generation of CD1 tetramers. The ability to generate CD1d tetramers has provided us with the opportunity of comparing a broad panel of CD1d binding compounds for their ability to stimulate iNKT cells.

A protocol for obtaining highly pure integral membrane proteins has been set up in the last two months in the lab of Paola Castagnoli in collaboration with the lab of Juri Rappsilber at IFOM.

We established protocols for the efficient in vitro transfection of primary dendritic cells based on viral and non-viral (nucleofection) methods (A. Mantei). These have been applied to modify DC surface molecules (Notch ligands) in the murine system (S. Vaddakadathou) or deliver antigens in both murine and human DC into either the MHC I or MHC II presentation pathways (A. Sattler, M. Dziubainau). For the latter we used a MHC II presentation pathway targeting vector originally published by Wang et al. 1999. Proof of principle studies for antigen delivery have been ...

This methodology incorporates a unique sample preparation procedure based on 96 well plate complex sample trypsinization, a nano-LC-based peptide separation coupled to MSMS analysis sequencing. This procedure led us to the identification of 600 phagosomal proteins from murine DC phagosomes.

Production of lentiviruses: Producing lentivirus in 293 FT Cells: Day 1: 4 x 106 293 FT in 10 ml DMEM (+10 % FCS (Tet free), +P/S) in a 10cm plate Day 2: transfection Day 3: change of medium Day 4: harvesting

Pathway analysis performed with Eu.Gene (Cavalieri et al., 2005), following the algorithm developed by Beltrame et al. (2009).

We are preparing a protocol for a phase I clinical trial in patients with advanced (stage III or IV) melanoma. Patients will receive a DC vaccination regimen followed by 3 adoptive transfers of autologous in vitro expanded T cells. This novel approach combines in vivo priming by the DC vaccine with passive immunotherapy by lymphocyte infusion. Patients will undergo surgical removal of a metastatic lesion and leukapheresis at the beginning of the trial to provide sources for tumor antigens as well as monocytes and T cells, which will be isolated from the leukaphe...

We have developed a technology to obtain from melanoma metastases melanin-free total tumor RNA, suitable for in vitro amplification and DC transfection.

Leukapheresis products from cancer patients was used as a starting material for monocyte enrichment by elutriation technology with ELUTRA® (Gambro). The monocytes were cultured for five days to immature DC (imDC´s) and subsequently transfected with different constructs encoding for the oncogene Her-2/neu and as control PSA using the technology of Amaxa biosystems or an adeno virus Her-2 full length construct. Levels of Her-2 expression was highest following transfection with 3 different truncated Transmembrane-extracellular constructs, in particular a Her-2 ra...