The company has now successfully developed a technique for the large-scale generation of DC in serum-free medium under GMP conditions, and are willing to share their know-how with Network Partners.

We are progressing steadily towards the delivery of a standardized method for the immuno-monitoring of DC vaccination trials using a finite number of defined tumor antigens. Our starting point was the method that we had developed previously to estimate blood frequencies of CD8 T lymphocytes against one defined antigenic peptide. It was based on an in vitro restimulation of blood lymphocytes with the peptide and growth factors over two weeks followed by detection with the appropriate HLA class I tetramer. In order to estimate frequencies of responding cells, these ...

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...

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...

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 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...

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 ...

Protocol for the documentation and the analysis of the interaction between RIG-I, a viral sensing protein, and NS1, an inhibitor of interferon production that is encoded by influenza A virus. This protocol has been made available.

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...

To generate a proteomic map of DCs (WP2) which includes quantitative information that can be used further to study signalling cascades (WP5) it is also necessary to optimize the SILAC labelling of DCs. This work was performed by Christian A. Luber (PhD student). First, we have set up a system for SILAC labelling of bone marrow derived murine dendritic cells (BM-DC). The principal method for generating BM-DC with GM-CSF was adapted from Lutz et al. (J Immunol Methods 1999, 223: 77-92). After some modifications of the protocol the FACS analysis of DC, which were gen...

For quantitative proteomic studies, our group recently described the SILAC (Stable Isotope Labelling by Amino Acids in Cell Culture) approach. In this method, independent populations of cells are grown in medium containing different non-radioactive isotope forms of essential amino acids (e.g. arginine and lysine). Because the cells cannot synthesize these amino acids, after some generations all proteins are labelled and therefore can be distinguished and quantified by the mass spectrometer.

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.

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 ...

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.

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...

A Phase II Study Testing the Activity and Safety in successfully ART-Treated Subjects Infected with HIV in Combination with ART Followed by ART Interruption. Study Design: The study design is identical to the Phase I study except that after the fourth dose patients will begin a drug treatment interruption period while receiving additional immunizations during that period. This provides an opportunity to assess the impact of the Arcelis therapy on viral load. Current status: Argos has received regulatory approval for this study and is actively accruing patients.

This is a Pilot Study (Phase I/II) testing the immunologic activity and safety of AGS-004, an autologous HIV immunotherapeutic, in HIV-infected adults on HAART. Study Design: Patients that are successfully treated with highly active anti-retroviral therapy (HAART) with no measurable viral load AND have a cryopreserved infectious plasma sample that was drawn immediately prior to initiation of HAART will be administered 4 monthly doses of the Arcelis product.

Draft templates for IMP have been generated.