We developed additional antigen-loading strategies for DC and obtained data from first experiments on which constructs lead to antigen presentation and which do not. On the one hand we compared lipofection of tumor-antigen-encoding RNA to RNA electroporation into DC, and found that electroporation results in antigen expression in all cells, while lipofection results in higher antigen expression only in a portion of the cells (other cells are negative). Interestingly, the priming capacity of MelanA/HLA-A2-specific autologous CD8+ T cells by lipofected DC was high...

Our group has obtained imaging data on the role of DALIS and related aggregates in antigen presentation and their interaction with the regulation of translation in response to pathogens. We have implemented in collaboration with lead coordinator Carl Figdor, the organization of the imaging core-facility of the DC-THERA NoE. Experimentally, we have been applying confocal microscopy to the study of DALIS and related aggregates in order to understand their role in antigen presentation and their interaction with the regulation of translation in response to pathogens....

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, including Langerhans cells, therefore influencing transfection efficiencies. We have assessed and obtained data on cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We showed that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell deat...

Data has been obtained from a Balb-neuT mice model to study new strategies for advanced breast cancer. A colony has been established of transgenic Balb-neuT mice (founders provided by Forni, Torino IT) that over-express the rat HER-2/neu oncogene under the MMTV promoter. These mice develop mammary carcinomas with later lung and liver metastases; the course of disease and the type of tumours that develop closely recapitulate many features of human breast cancer. A collaborative study has commenced between Austyn and Cerundolo to use this model to study new strategi...

We have developed structural, kinetic and functional data to study the binding of ligands to CD1 molecules and activation of CD1d restricted iNKT cells. The results of these studies have led to: i) The development of protocols to refold in vitro CD1 molecules, which were used for the generation of CD1 tetramers. 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. ii) Identification of a novel population of NKT cells, which does not express the ca...

Our group has generated pathway data from human DC treated with pathogen derivatives.

We obtained data on the biological activity of an LPS-like molecule extracted from the freshwater cyanobacterium Oscillatoria Planktothrix FP1 that we named CyP. We found that CyP acts as a potent and selective antagonist of bacterial LPS. CyP did not induce any detectable response in human DCs and competitively inhibited LPS binding to the extracellular domain of TLR4. Addition of CyP together with LPS completely inhibited both MyD88- and TRIF-dependent pathways and suppressed the whole LPS-induced gene transcription program. CyP was effective in protecting mice ...

Data has been obtained on the activation of human DC by Toll like receptor ligands (TLR-L) that modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells. DC derived soluble factors further increase CD1d-restricted iNKT cell activation, as defined by IFN-alpha secretion. Finally, using soluble tetrameric iNKT TCR as a staining reagent we demonstrate specific up-regulation of CD1d-bound ligands upon TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of DC resulting in iNK...

We have obtained data from a study where we addressed a model antigen to exosomes secreted by tumor cells in vivo, to allow capture of antigen-bearing exosomes by DCs and cross-presentation of the antigen. For this purpose, the model antigen OVA was fused to the C1C2 exosome-binding domain of MFG-E8/lactadherin. Tumors secreting the exosome-bound antigen grow slower than tumors secreting soluble OVA, because they induce activation of OVA-specific CD8+ T cells into killer cells. In addition, inducing in vivo secretion of the C1C2-coupled antigen by DNA vaccination i...

We obtained data on MHC II transport during human monocyte-derived DC maturation. We have published that the E3-ubiquitin ligase MARCH I is responsible for peptide-loaded MHC II ubiquitination and their subsequent internalization in immature DC.s The ligase MARCH I has been shown to be down-regulated upon activation. (De gassart et al, PNAS 2008). We will also investigate further the role of the ubiquitin ligases of the MARCH family in antigen processing, in particular we will focus on cross-presentation and CD1a molecule transport.

(This trial is expected for 2009 and data will become available.)

In 2008 we have started a trial in patients with VIN3 disease (similar to the Phase I/II clinical trial on the use of long peptides plus adjuvant for end-stage cervical cancer or VIN patients), in which we are comparing the efficacy of this vaccine with or without local application at the vaccination site of the TLR ligand imiquimod (Aldara). Results and data will become available.

Our lab obtained first data from a new clinical trial of a DC vaccine administered into irradiated tumours in RCC. Three patients were enrolled and DC vaccine was produced. However only one patient received the vaccine due to cancer progression in the other two patients. The DC were labelled with 111In. The following patients will receive DC labelled with Endorem and the vaccine will be imaged with MRI. The patient treated is in complete remission 6 months after treatment.

We obtained data from a trial in small cohorts of patients (3-9) who were vaccinated with differently composed DC-vaccines: DC loaded with a single MAA-derived peptide (MAGE-3.A1 or -A2 or Na17.A2) or pulsed with 2 to 3 MAA-derived peptides (combinations of MAGE-3.A2, MAGE-C2.A2 or Na17.A2) or G4-DC loaded with 8 different peptides. Our experiences can be summarized as follows: (1) preparation of an autologous dendritic cell based vaccine was feasible for every patient enrolled in our studies; (2) we did not observe vaccination related toxicities beside mild...

We have continued recruitment of patients and obtained first data in the two clinical trials of active vaccination in metastatic melanoma. Structural works that have been performed to up-grade the GMP facility for conduction of phase III clinical trials has limited the number of patients recruited. Nonetheless, we were able to complete the vaccination schedule in 4 patients and to perform additional injections in disease free patients, in patients with stable disease or with slowly progressive disease. We could not recruit new patients because the generation in GM...

We have further data available on a clinical trial on vaccination of B-CLL patients with autologous, apoptotic, leukemic cells (Apo-DC). Cohort 1 with the vaccine alone and cohort 2 (5 patients) combining the vaccine together with GM-CSF as an adjuvant has finished accrual and all patients in these two cohorts have been monitored for 52 weeks. The clinical and immune monitoring data is being currently analyzed. The third cohort consisting of the vaccine and GM-CSF administered after a single dose of cyclophosphamide (300 mg/ sq. meter) is currently being accrued...

Our lab obtained data on the ability of NK cell IFN-gamma production to predict long term survival in advanced GISTs treated with IM. Background: Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for imatinib mesylate (IM) (Gleevec®, Novartis), a specific inhibitor of these tyrosine kinase receptors. Predictive factors for the response to IM pertain to the intrinsic features of GIST tumor cells but not to the host. Besides its direct antiproliferative activity on tumor cells, I...

A phase I/II study of immunization of HIV infected individuals stable under HAART has been initiated which will provide us with immunological data. These patients are being vaccinated with cytokine cocktail matured DCs electroporated with mRNA encoding Tat, Rev and Nef. The cDNA’s encoding these early expressed HIV antigens have been human codon optimized and modified with lysosomal targeting sequences. Four vaccines separated by 4 weeks. Two weeks after the 4th vaccine, an analytical HAART interruption was started. So far, 17 patients have been included in t...

During the previous period we obtained data from our continued pilot clinical trials of DC-based immunotherapy of melanoma patients. After an initial cohort of patients treated with monocyte-derived DCs matured with inflammatory cytokines and subsequently electroporated with mRNA encoding 6 tumor-associated antigens, we persued this trial in a next cohort where DC-based vaccines were combined with low dose IFN-alfa (3 x 10e6 U per wk). In a significantly higher number of patients we observed vaccine-induced depigmentation and vitiligo. Further improvement and ca...

Summary of the completed RCC clinical study and the data obtained: RCC subjects were deficient in T cell IFN-gamma and IL-2 production pre-vaccination. The IL-2 defect was corrected in 7/12 patients post-vaccination. Vaccination resulted in an increase in tumor antigen-specific T cells in 8/12 patients. 7/12 subjects had a response to more than one antigen post-vaccination. Median overall survival was ~25 months compared to ~11 months for historical controls (confirmed by matched pair analysis).