Anticancer activity of oncolytic adenovirus vector armed with IFN-alpha and ADP is enhanced by pharmacologically controlled expression of TRAIL

Abstract

We have previously described oncolytic adenovirus (Ad) vectors KD3 and KD3-interferon (IFN) that were rendered cancer-specific by mutations in the E1A region of Ad; these mutations abolish binding of E1A proteins to p300/CBP and pRB. The antitumor activity of the vectors was enhanced by overexpression of the Adenovirus Death Protein (ADP, E3-11.6K) and by replication-linked expression of IFN-alpha. We hypothesized that the anticancer efficacy of the KD3-IFN vector could be further improved by expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). E1-deleted Ad vectors were constructed carrying reporter genes for enhanced green fluorescent protein or secreted placental alkaline phosphatase (SEAP) and a therapeutic gene for TRAIL under control of the TetON system. Expression of the genes was increased in the presence of a helper virus and the inducer doxycycline such that up to 231-fold activation of expression for the TetON-SEAP vector was obtained. Coinfection with TetON-TRAIL augmented oncolytic activity of KD3 and KD3-IFN in vitro. Induction of TRAIL expression did not reduce the yield of progeny virus. Combination of TetON-TRAIL and KD3-IFN produced superior antitumor activity in vivo as compared with either vector alone demonstrating the efficacy of a four-pronged cancer gene therapy approach, which includes Ad oncolysis, ADP overexpression, IFN-alpha-mediated immunotherapy, and pharmacologically controlled TRAIL activity.

Figure 1

Schematics of vector genomes. Open arrows, early Ad regions. Closed arrows, late Ad regions. Gray arrows or boxes, inserted transgenes. Closed boxes, inverted terminal repeats (ITR). Short arrow in an open box, minimal CMV promoter. TRE, tetracycline-response element; rtetR, reverse tetracycline repressor; tetO, tetracycline operator; rtTA, reverse tetracycline transcriptional activator; pA, polyadenylation signals. (a) KD3 and KD3–IFN are conditionally replicative Ad vectors carrying two small deletions in the E1A region rendering virus replication cancer-specific. The vectors overexpress ADP for improved spread. KD3–IFN expresses human IFN-α2b strictly late in infection for increased anticancer activity and reduced hepatotoxicity of the vector. (b) TetON–TRAIL, TetON–EGFP and TetON–SEAP are replication-defective Ad vectors carrying TRAIL, EGFP or SEAP expression cassettes, respectively, in place of the E1 region deletion. The CMV promoter in the expression cassette drives expression of rtTA which consists of rtetR fused to VP16, a transcriptional activator derived from herpes simplex virus. rtTa binds to tetO in the presence of Dox, activating a minimal CMV promoter and leading to transgene expression. CMV, cytomegalovirus. EGFP, enhanced green fluorescent protein; IFN, interferon; SEAP, secreted placental alkaline phosphatase; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 2

Coinfection with oncolytic Ad allows for regulatable expression of SEAP from the RD Ad TetON–SEAP. (a) Hep3B, (b) A549, (c) DLD-1 and (d) 293 cells were infected with TetON–SEAP, TetON–SEAP+KD3 or TetON–SEAP+KD3–IFN at an MOI of 10PFU per cell for each virus in the presence or absence of Dox. SEAP enzymatic activity in the medium was determined at various time points. MOI, multiplicity of infection; PFU, plaque forming unit; RD, replication deficient; SEAP, secreted placental alkaline phosphatase.

Figure 3

TetON–TRAIL expresses TRAIL. (a) Detection of TRAIL expression by TetON–TRAIL after coinfection with RC Ad in the presence of Dox. Hep3B cells were infected with the indicated vectors at an MOI of 50PFU per cell for each virus in the presence or absence of Dox. TRAIL expression in the cells at day 2 postinfection was detected by western blotting. (b) The functional activity of TRAIL and EGFP is regulated by Dox in cells coinfected with TetON–TRAIL or TetON–EGFP and helper RC Ad. Hep3B cells were coinfected with TetON–TRAIL and KD3 or with TetON–EGFP and KD3 at an MOI of 10PFU per cell in the presence or absence of Dox. Phase-contrast or fluorescence microphotographs were taken at indicated time points. Original magnification × 100. Arrows indicate the apoptotic morphology of cells. EGFP, enhanced green fluorescent protein; MOI, multiplicity of infection; PFU, plaque forming unit; RC, replication competent; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 4

TRAIL enhances the oncolytic effect of tumor-specific, RC Ad in cancer cell lines. (a) Hep3B and (b) DLD-1 cells were infected with KD3, TetON–TRAIL+KD3, TetON–EGFP+KD3, KD3–IFN, TetON–TRAIL+KD3–IFN or TetON–EGFP+KD3–IFN at an MOI of 10PFU per cell for each virus in the presence or absence of Dox. Cell viability was quantified at day 3 (Hep3B) or day 2 (DLD-1) postinfection. EGFP, enhanced green fluorescent protein; IFN, interferon; MOI, multiplicity of infection; PFU, plaque forming unit; RC, replication competent; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 5

Influence of TRAIL on spread and extracellular yields of oncolytic Ad. (a) High level of TRAIL expression impairs TetON–TRAIL spread in 293 cells; low basal expression improves the spread. 293 cells were infected with TetON–TRAIL or TetON–EGFP at various MOI. Transgene expression was induced by Dox. Cell viability was quantified at day 6 postinfection. (b) Expression of TRAIL increases the extracellular titers of the tumor-specific oncolytic Ad in Hep3B cells. Cells were coinfected with TetON–TRAIL and KD3, or TetON–EGFP and KD3, at an MOI of 10PFU per cell. Transgene expression was induced by addition of Dox to the medium at 0 or 24 h postinfection. Virus titers were determined in the medium at 48 h postinfection. EGFP, enhanced green fluorescent protein; MOI, multiplicity of infection; PFU, plaque forming unit; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 6

Antitumor activity of KD3–IFN combined with regulatable expression of TRAIL. (a) Expression of TRAIL increases the antitumor efficacy of the cancer-specific Ad armed with ADP and IFN-α. Established subcutaneous Hep3B tumors were injected intratumorally with PBS (n = 5), 1 × 10⁹PFU of KD3–IFN (n = 11), 1 × 10⁸PFU of TetON–TRAIL (n = 5) or 1 × 10⁹PFU of KD3–IFN plus 1 × 10⁸PFU of TetON–TRAIL (n = 11). The injections were repeated with 1-day intervals for a total of five injections. Mice injected with TetON–TRAIL or TetON–TRAIL+KD3–IFN received Dox with the drinking water. (b) For animals from (a) Kaplan–Meier survival curves were plotted. ADP, Adenovirus Death Protein; IFN, interferon; PFU, plaque forming unit; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.