Transductionally Redirected /Transcriptionally Restricte

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Adenovirus vectors are among the most popular vehicles for cancer gene therapy protocols. However, adenovirus infect cells via the Coxsackie and Adenovirus Receptor, which is often more extensively expressed on normal cells then on tumors. We utilized bioluminescent imaging following systemic Ad.CMVfLuc administration to monitor adenovirus transductional "untargeting" and "retargeting" by sCAREGF. sCAR-EGF is a bi-specific retargeting molecule containing the soluble portion of CAR linked to epidermal growth factor. Noninvasive optical imaging demonstrates that systemically injected [Ad.CMVfLuc] [sCAR-EGF] complexes have reduced ability to infect liver, but can infect EGF receptor-positive xenografts. COX-2 is not expressed in most normal tissues, but is overexpressed in many tumors. We used noninvasive optical imaging to examine fLuc expression following intratumoral injection of Ad.COX2fLuc (an adenovirus expressing luciferase from the COX-2 promoter). No expression occurs in liver. In contrast, expression occurs in COX-2 positive tumors. Colorectal cancer (CRC) is a leading cause of cancer death. CRC frequently metastasizes to liver. CRC liver metastases often over-express both carcinoembryonic antigen (CEA) and COX-2. We will combine our experience - both with transductional "untargeting" and "retargeting", and with restricted COX-2 gene expression - to develop effective therapies for CRC hepatic metastases. We will establish xenograft CRC models of liver metastasis. We will create Ad.COX2.fLucTK.COX2, an adenovirus that expresses a fusion protein containing both firefly luciferase and HSV1-thymidine kinase (fLucTK). CRC tumor-restricted fLucTK expression will result from regulation by both the COX-2 promoter and the COX-2 mRNA 3' untranslated region. Transductional liver untargeting and Ad.COX2.fLuTK.COX2 transductional retargeting to CRC xenograft metastases will be optimized with sCAR-f-alphaCEA, a recombinant molecule containing soluble CAR, the phage T4 fibritin trimerization domain and a single-chain anti-CEA antibody. Combined sCAR-f-alphaCEA transductional liver untargeting, transductional tumor retargeting and COX-2 restricted expression should optimize fLucTK expression in CRC hepatic metastases. HSV1-TK/ganciclovir therapy should then effectively eradicate CRC metastases, with minimal side effects. We will employ bioluminescent imaging to monitor restricted fLucTK expression, bioluminescent imaging from xenografts marked with Renilla luciferase and 124I-antiCEA minibody/microPET imaging to monitor tumor burden during HSV1- TK/GCV therapy, and FDG and FLT/microPET to monitor early therapeutic responses. Our goal is to optimize this therapeutic protocol for CRC hepatic metastases, in apreclinical model of human disease.