Tumor-Specific, Replication-Competent Adenovirus Vectors Overexpressing the Adenovirus Death Protein

Abstract

We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl 1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G 0 to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus,dl 1101/1107, or wild-type Ad. They are also more efficient than dl 1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1−E3−replication-defective vector expressing β-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10−4 PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl 1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.