| Gastro-intestinal tumors. In collaboration with the Departments of Gastro-enterology (Heideman, Craanen) and Molecular Pathology (Snijders), the expression of antigens on gastric carcinoma samples was investigated. The receptor for adenovirus (Coxsackie Adenovirus Receptor; CAR) was detectable on tumor cells, but the expression level was relatively low. The tumor antigen EPCAM was highly expressed on malignant and pre-malignant lesions, but not on normal gastric mucosa. Furthermore, the avb3 or avb5 ntegrins were highly expressed on malignant cells and not on normal tissue. The c-MET (receptor for Hepatocyte Growth Factor/Scatter Factor) receptor is predominantly expressed on malignant cells. Using reporter genes, targeting experiments have revealed that the EPCAM antigen could be used for targeting of adenoviral vectors with an immunoconjugate. Dr. Victor Krasnykh / Prof. Curiel have inserted the RGD sequence in the fiberknob of an adenovirus. The RGD sequence binds to the avb3 or avb5 integrins. This genetically modified adenoviral vector could very efficiently transduce gastric carcinoma cells, but since the CAR-dependent adhesion is not ablated the vectors also bound to normal gastric cells. These two new targeting moieties will be further explored for specific transduction of gastric carcinoma cells. Brain tumors. Gene therapy of brain tumors is usually achieved by direct injection of vectors into brain tumors. The limitation of this approach is that gene expression is restricted to a small area around the needle tract. In collaboration with the Department of Pediatrics (Institut Gustave Roussy), Department of Neurosurgery (Dr.C.M.F. Dirven, Prof.dr. Vandertop), and the Department of Radiobiology (Dr. P. Sminia), two preclinical models were established; a rat model for intra-arterial delivery of adenoviral vectors towards intracranial tumors, and a spheroid model for studying the penetration of adenoviral vectors in solid tumors. Analysis of tumor samples of patients have revealed that the EGF receptor and the avb3 or avb5 integrins are expressed both on tumor vascular endothelium cells and tumor cells, and not on their normal counterparts. However, there exists variability from patient to patient with regard to the expression of these antigens. Preliminary results have indicated that a combination of genetic targeting (RGD sequence in fiberknob) and immunological targeting (EGFR/anti-knob) results in an additive enhancement of transduction of the glioblastoma cells. These two targeting moieties will be tested in the rat model for glioblastoma. Addition of titrated doses of adenoviral particles in medium to spheroids have revealed that only the outer cell layers are tranduced. The spheroid model is ideally suited to investigate whether manipulations, such as irradiation, of the tumor cells will enhance the penetration of the viral particles. Furthermore, it will be used to investigate the diffusion of enzymes and/or drugs in enzyme/prodrug strategies and the penetration of replicative adenoviruses. Bone tumors. In 1999 a new project was started to investigate gene therapy of osteosarcoma. Since it is difficult to inject adenoviral vectors into this bony tumor and CAR expression is low on tumor cells, this project is aiming at intravascular delivery and targeting of adenoviral vectors. Tumor immunology. Dendritic cells (DCs) have a low expression of the CAR receptor and can only be transfected with adenoviral vectors at high MOI (>1000). Using a bispecific antibody (anti-CD40/anti-fiberknob) adenoviral vectors were targeted to DCs. This resulted in specific transduction of CD40 positive DCs and activation of the DCs (maturation, production of IL-12 and optimal functioning of DCs) by triggering the CD40 molecule. In a skin explant model, total injection of GM-CSF recruited DCs and these DCs could be targeted in situ using adenoviral vectors and bispecific antibody. The transduced DCs were still able to migrate in this skin explant model. |
