23. T Cell Carriers for Gene Delivery to Tumors
Authors: Maaike Everts, Sang Ae Kim-Park, Long P. Le, Paul N. Reynolds, William E. Grizzle, David T. Curiel
Journal: Molecular Therapy (2004)

Recurrent colorectal adenocarcinoma can be treated by surgery and chemotherapy, but effects on length of survival time are generally modest. Therefore, identification of new therapeutic modalities is of great importance to improve the survival of these patients. Adenoviral gene delivery systems have shown promise as cancer therapeutics, but insufficient or non-selective transduction of tumor cells remains an issue. Targeting adenoviral vectors (Ads) to tumor-associated antigens, such as carcinoembryonic antigen (CEA, which is overexpressed in colon carcinomas), may overcome this problem. Ads can be targeted using %targeting adapter molecules', such as sCAR-scFv fusion proteins. One part of these fusion proteins consists of the ectodomain of the Coxsackie and Adenovirus Receptor (sCAR, the primary receptor for Ad), which binds to the adenoviral particle. The other part consists of a CEA recognizing modality, such as a single chain antibody (scFv). In vitro, these fusion proteins have already shown great promise for targeting Ads to cancer cells. However, availability of animal models in which CEA is expressed is limited. This has hampered the subsequent in vivo characterization of these sCAR-scFv fusion proteins. We hypothesized that we could overcome these problems by using a transgenic hCAR mouse (a kind gift of Dr. S. Petterson, Sweden), in combination with Ads encoding CEA under transcriptional control of the endothelial specific flt-1 promoter to induce CEA in the pulmonary vasculature of these mice. In this manner we could define key Ad targeting issues, independent of specific tumor biology factors. First, Ad biodistribution in hCAR mice was determined by injecting a GFP labeled Ad (AdIXEGFP) in the inferior vena cava. Compared to wild type mice, hCAR mice showed an increased uptake of AdIXEGFP particles in the lungs. Second, transgene biodistribution was determined by administering a luciferase-encoding Ad (AdCMVLuc) in the tail vein. As expected from the change in biodistribution, hCAR mice showed an increase in luciferase activity in the lungs. However, luciferase expression in the liver, the main organ of ectopic transgene expression after systemic Ad administration, was high as well. To increase selectivity of transgene expression in the lungs, we employed the endothelial specific promoter flt-1. This resulted in significantly reduced liver luciferase expression, thereby increasing the lung/liver ratio by several orders of magnitude. Finally, AdfltCEA was administered to study cellular distribution of CEA using immunohistochemical techniques, showing that CEA transgene expression in the lungs was largely confined to pulmonary endothelial cells. Thus, selective expression of CEA in the pulmonary vasculature by use of the here-described in vivo system provides a valuable tool for transductional targeting studies. The targeting adapter molecule sCAR-scFv, recognizing CEA, can now be tested for its targeting potential in this %transient transgenic' mouse model. This is of vital importance before translation to a clinical setting for the treatment of colon carcinoma can be endeavored.