Gene Therapy (2001) 8, 969–976  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin- targeted adenoviral vectors JG Wesseling 1 , PJ Bosma 1 , V Krasnykh 3 , EA Kashentseva 3 , JL Blackwell 3 , PN Reynolds 3 , H Li 3 , M Parameshwar 3 , SM Vickers 4 , EM Jaffee 5 , K Huibregtse 2 , DT Curiel 3 and I Dmitriev 3 1 Department of Experimental Hepatology, 2 Department of Gastroenterology and Liver Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 3 Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery and the Gene Therapy Center and 4 Department of General Surgery, School of Medicine, University of Alabama at Birmingham, Birmingham, AL; and 5 Department Of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA In this study we analyzed two ways of retargeting of Ad- vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carci- noma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispe- cific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) gen- etically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency Keywords: pancreatic carcinoma; primary tumor cells; adenovirus vector; targeting; gene therapy Introduction Pancreatic cancer is highly aggressive and ranks fifth among malignancy-associated deaths. Prognosis remains dismal because diagnosis of pancreatic cancer is made late in the clinical course of the disease. Currently, there is no effective treatment for this disease: resection is only available to a small fraction of patients presenting with locally confined tumor. 1 Chemotherapy and radiation also have limited effects on patient survival. Adjuvant combined radiochemotherapy might potentially improve survival and can be considered in unresectable, locally advanced disease. However, the role of chemotherapy in advanced disease is exclusively palliative. 1 Therefore, development of new therapeutic modalities such as gene therapy are necessary to improve patient outcome and serve as a more effective treatment for pancreatic cancer. Adenoviral vectors have been used for both in vitro and in vivo gene delivery of pancreatic cancer, mainly because Correspondence: DT Curiel, Division of Human Gene Therapy, Dept of Medicine, Pathology, Surgery and Gene Therapy Center, The University of Alabama at Birmingham, 1824 6th Avenue South, 620 Lurleen Wallace Tumor Institute, Birmingham AL 35294–3300, USA Received 13 November 2000; accepted 9 March 2001 to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both av integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-con- taining peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR- independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma. Gene Therapy (2001) 8, 969–976. of their ability to infect both dividing pancreatic cancer cells, as well as nondividing tumor cells. Another advan- tage is that the techniques to produce high-titered prep- arations of adenovirus vectors are relatively simple. Fur- thermore, phase I clinical trials employing adenovirus vectors have been started already for pancreatic cancer. 2 As observed for other tumor tissue types, a major concern associated with using adenovirus vectors in pancreatic cancer is the relatively limited infection efficiencies achi- eved in vitro. 3 Furthermore, in vivo gene delivery may be limited by other factors, such as vector’s access to target cells through local dissemination or through penetration of vessel walls. Studies on adenoviral entry into host cells have revealed that two cell surface events, attachment and internalization, are required for an adenovirus to enter a cell. 4 The viral fiber protein will first attach to the CAR (Coxsackie and adenovirus receptor) on the surface of a host cell. 5 The virion then enters the cell through the interaction of its penton base with the avb3 and avb5 integrins on the host cell surface. 4 Expression of these cell surface markers and their correlation with the efficiency of adenovirus-mediated gene transfer have revealed that the presence of integrins avb3, 6 avb5 7 and CAR 8,9 are important for an efficient gene transfer and efficacy of