RESEARCH ARTICLE Noninvasive dual modality in vivo monitoring of the persistence and potency of a tumor targeted conditionally replicating adenovirus A Kanerva 1,2,3 , KR Zinn 4 , K-W Peng 5 , T Ranki 1 , L Kangasniemi 1 , TR Chaudhuri 4 , RA Desmond 6 , M Wang 7 , K Takayama 8 , T Hakkarainen 9 , H Alfthan 3 , U-H Stenman 3 , DT Curiel 7 and A Hemminki 1,2 1 Cancer Gene Therapy Group, Rational Drug Design, Biomedicum Helsinki, University of Helsinki, Finland; 2 Department of Oncology, Helsinki University Central Hospital, Helsinki, Finland; 3 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland; 4 Department of Radiology, University of Alabama at Birmingham, Birmingham, AL, USA; 5 Molecular Medicine Program, Mayo Clinic, Rochester, MN, USA; 6 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA; 7 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, USA; 8 Research Institute for Diseases of the Chest, Kyushu University, Japan; and 9 Department of Biotechnology and Molecular Medicine, AI Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland In clinical trials with cancer patients, the safety of condition- ally replicating adenoviruses (CRAds) has been good. However, marginal data are available on the persistence or antitumor efficacy of these agents. The oncolytic potency of CRAds is determined by their capacity for entering target cells. Consequently, we constructed a retargeted CRAd featuring a secreted marker protein, soluble human carci- noembryogenic antigen (hCEA), which can be measured in growth medium or plasma. We found that virus replication closely correlated with hCEA secretion both in vitro and in vivo. Further, antitumor efficacy and the persistence of the virus could be deduced from plasma hCEA levels. Finally, using in vivo bioluminescence imaging, we were able to detect effective tumor cell killing by the virus, which led to enhanced therapeutic efficacy. Gene Therapy (2005) 12, 87–94. doi:10.1038/sj.gt.3302387 Published online 23 September 2004 Keywords: adenovirus; ovarian neoplasms; virus replication; imaging Introduction Conditionally replicating adenoviruses (CRAds) repre- sent a promising approach for treating cancer refractory to traditional therapies. 1 Recently, it has been demon- strated that the oncolytic potency of virotherapy with replicating agents is partly determined by their cap- ability of infecting target cells. 2,3 Most adenovirus vectors and CRAds are based on serotype 5 (Ad5), which binds to the coxsackie-adenovirus receptor (CAR). Unfortu- nately, recent data suggest variable CAR expression on ovarian and some other cancer cells, which could hinder CRAd-mediated oncolysis in these cancer cells. 4 There- fore, methods to circumvent CAR deficiency have been evaluated. Previously, we evaluated a serotype chimeric Ad5/3 adenovirus possessing the receptor-binding fiber knob domain of Ad3 in the Ad5 capsid, which retargeted the virus to the Ad3 receptor highly expressed on ovarian and other cancer cells. 5,6 Recently, it has been suggested that there is a common receptor for subgroup B adenoviruses, which Ad3 also belongs to. 7 Specifically, the CD46 membrane cofactor protein has been pro- posed. 8,9 In contrast, a recent publication suggests that Ad3 does not use CD46 as an attachment receptor. 9 CD46 is a member of a family of glycoproteins acting as regulators of complement activation, and therefore prevents spontaneous activation of complement on autologous cells. Interestingly, also CD80 and CD86 co- stimulatory molecules have been proposed as candidate Ad3 receptors. 10 As Ad5/3 chimerism increased gene delivery to tumor but not normal cells, we created an Ad3 receptor retargeted CRAd, Ad5/3-D24. 3 This oncolytic virus has a 24-bp deletion in E1A, resulting in a mutant E1A protein unable to bind in the Rb protein, which normally allows S-phase entry, needed for virus replication. Therefore, Ad5/3-D24 replicates in cancer cells inactive in the Rb/p16 pathway, 11 including most if not all human cancer cells. 12 Ad5/3-D24 displayed enhanced oncolytic potency and therapeutic efficacy in ovarian cancer models as compared to an otherwise isogenic CRAd, but with the wild-type fibers. Although the safety data with CRAds in clinical trials have been excellent, not much is known about persis- tence or antitumor activity, as there is a paucity of means Received 21 November 2003; accepted 3 August 2004; published online 23 September 2004 Correspondence: Dr A Hemminki, Rational Drug Design, Biomedicum Helsinki, Department of Oncology, Helsinki University Central Hospital, PO Box 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland Gene Therapy (2005) 12, 87–94 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt