PDF file - 2.2MB, (A) Cell monolayers were infected with 100 viral paticles /cell (VP/cell) and 24 hours later cells were analyzed by flow-cytometry measuring Annexin-V expression. Results are presented as percentage of positive cells relative to Mock uninfected cells. **, P<0.01. (B) CD40 expression is shown in brown in A549 and EJ tumors. Pictures were taken with an Axioplan Microscope equipped with a camera Axiocam at a magnification of 10x. (C) Tumors from nude mice bearing either A549 tumors (left panel) or EJ tumors (right panel) injected with viruses were analyzed for viral load. Quantitative PCR was performed for E4 sequence and values were normalized to human beta-actin gene.. The real-time PCR conditions were as described in (31). All samples were tested in duplicate. Results are presented as mean from all mice +SEM. **, P<0.01.
ARTICLE ABSTRACT
Oncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly immunogenic and not subject to tolerance. Although oncolysis releases tumor epitopes and provides costimulatory danger signals, arming the virus with immunostimulatory molecules can further improve efficacy. CD40 ligand (CD40L, CD154) induces apoptosis of tumor cells and triggers several immune mechanisms, including a T-helper type 1 (TH1) response, which leads to activation of cytotoxic T cells and reduction of immunosuppression. In this study, we constructed a novel oncolytic adenovirus, Ad5/3-hTERT-E1A-hCD40L, which features a chimeric Ad5/3 capsid for enhanced tumor transduction, a human telomerase reverse transcriptase (hTERT) promoter for tumor selectivity, and human CD40L for increased efficacy. Ad5/3-hTERT-E1A-hCD40L significantly inhibited tumor growth in vivo via oncolytic and apoptotic effects, and (Ad5/3-hTERT-E1A-hCD40L)–mediated oncolysis resulted in enhanced calreticulin exposure and HMGB1 and ATP release, which were suggestive of immunogenicity. In two syngeneic mouse models, murine CD40L induced recruitment and activation of antigen-presenting cells, leading to increased interleukin-12 production in splenocytes. This effect was associated with induction of the TH1 cytokines IFN-γ, RANTES, and TNF-α. Tumors treated with Ad5/3-CMV-mCD40L also displayed an enhanced presence of macrophages and cytotoxic CD8+ T cells but not B cells. Together, our findings show that adenoviruses coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis, induction of T-cell responses, and upregulation of TH1 cytokines. Cancer Res; 72(9); 2327–38. ©2012 AACR.
