Simple SummaryOncolytic adenoviruses are genetically engineered to selectively replicate within cancer cells by exploiting their unique characteristics while sparing normal cells. However, adenoviruses generally require the coxsackie and adenovirus receptor (CAR) to enter the cells. This study investigated the activity of an oncolytic adenovirus in tumors with low CAR expression. The results showed that 58% and 33% of the tumors treated with liposome-encapsulated virus and viruses without liposome encapsulation achieved complete remission, respectively. Microscopic tissue analysis shows that an increased inflammatory infiltrate inside the tumor microenvironment is key to achieving effective therapeutic results in tumors independent of CAR expression. In the bilateral tumor model, both unencapsulated and encapsulated viruses reduced local tumor growth. However, only encapsulated virus reduced growth in distant tumors. Encapsulated adenovirus demonstrated an increased inflammatory infiltrate, including CD-8 and NK cells in both the treated and untreated tumors, whereas unencapsulated virus demonstrated limited inflammatory infiltrate in both tumors.AbstractThis study evaluated the in vivo therapeutic efficacy of oncolytic serotype 5 adenovirus TAV255 in CAR-deficient tumors. In vitro experiments were performed with cell lines that expressed different levels of CAR (HEK293, A549, CT26, 4T1, and MCF-7). Low CAR cells, such as CT26, were poorly transduced by Ad in vitro unless the adenovirus was encapsulated in liposomes. However, the CT26 tumor in an immune-competent mouse model responded to the unencapsulated TAV255; 33% of the tumors were induced into complete remission, and mice with complete remission rejected the rechallenge with cancer cell injection. Encapsulation of TAV255 improves its therapeutic efficacy by transducing more CT26 cells, as expected from in vitro results. In a bilateral tumor model, nonencapsulated TAV255 reduced the growth rate of the locally treated tumors but had no effect on the growth rate of the distant tumor site. Conversely, encapsulated TAV255-infected CT26 induced a delayed growth rate of both the primary injected tumor and the distant tumor, consistent with a robust immune response. In vivo, intratumorally injected unencapsulated adenoviruses infect CAR-negative cells with only limited efficiency. However, unencapsulated adenoviruses robustly inhibit the growth of CAR-deficient tumors, an effect that constitutes an ‘in situ vaccination’ by stimulating cytotoxic T cells.