Antitumor immune responses mediated by adenoviral GDEPT using nitroreductase/CB1954 is enhanced by high-level coexpression of heat shock protein 70.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising approach to local management of cancer through targeted chemotherapy. Killing localized tumors by GDEPT in a manner that induces strong antitumor cellular immune responses might improve local management and allow benefit in disseminated cancer. Here we evaluated the combination of nitroreductase (NTR)/CB1954 GDEPT with high-level expression of heat shock protein 70 (HSP70, a stress protein that can shuttle cytosolic peptides into antigen-presenting cells) for induction of antitumor immunity using adenovirus gene delivery in an aggressive and nonimmunogenic BALB/c syngeneic 4T1 breast cancer model. The mechanism of cell death and spectrum of stress proteins induced are likely to be important determinants of the resulting immune responses. We showed that NTR/CB1954 treatment of 4T1 cells gave both apoptotic and nonapoptotic killing. In vivo killing of 4T1 cells expressing NTR gave weak antitumor immunity and very limited induction of stress proteins including HSP70. High-level coexpression of HSP70 during NTR/CB1954-mediated killing of 4T1 cells in vivo gave much greater protection from tumor challenge (67% long-term survivors compared to 17%) and induced 4T1-specific cytotoxic T-cell responses. The enhancement of antitumor responses resulting from HSP70 coexpression was similar to that conferred by coexpression of GM-CSF.

Optimization of a synthetic beta-catenin-dependent promoter for tumor-specific cancer gene therapy.

We recently published the construction and evaluation of a beta-catenin-dependent, highly active promoter, CTP1, and its possible application for the treatment of colorectal cancer using gene-directed enzyme prodrug therapy with adenoviral (Ad) vectors. Alternative Ad-based approaches such as tumor-specific, replication-competent vectors and/or exploiting therapeutic gene products with intrinsic toxic activity, such as gibbon ape leukemia virus fusogenic membrane glycoprotein, diphtheria toxin A (DTA), and ricin, would demand a very tightly regulated promoter to avoid breakthrough replication and toxicity in nontumor tissue and Ad producer cell lines. In this study we optimized the activity/specificity profile of the synthetic beta-catenin-dependent promoter by varying its basal promoter, the number of Tcf binding sites, and the distance between these and the basal promoter. The optimal promoter, CTP4, showed virtually undetectable expression in cells with normal beta-catenin regulation but high level expression in cells deregulated for beta-catenin. Using CTP4 we were able to generate, for the first time to our knowledge, an Ad vector expressing fully active wild-type DTA without the need for time-consuming and cumbersome production systems. CTP4 should be the promoter of choice for Ad-based gene therapies of tumors deregulated for beta-catenin. We provide preliminary evidence that these may include prostate and ovarian as well as colorectal cancer.

An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated wnt signaling.

We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3-11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear beta-catenin.