Potent and long‐term antiangiogenic efficacy mediated by FP3‐expressing oncolytic adenovirus

Abstract

Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.

Combined therapy with oncolytic adenoviruses encoding TRAIL and IL-12 genes markedly suppressed human hepatocellular carcinoma both in vitro and in an orthotopic transplanted mouse model

“…The resultant homologous plasmid DNA recombinants were further digested with Pac I and transfected into HEK293 cells to generate Ad-ΔB/TRAIL and Ad-ΔB/IL-12. The three generated OAds (Ad-ΔB, Ad-ΔB/TRAIL, and Ad-ΔB/IL-12) were further propagated in HEK293 cells, purified by CsCl gradient density purification method, and then dissolved in storage buffer (10 m M Tris, 4 % sucrose, 2 m M MgCl 2 ) and stored at−80 °C until use [ 29 , 30 ]. The number of viral particle (Vp) for each virus was calculated from measurements of optical density at 260 nm (OD 260 ), where one absorbency unit is equivalent to 1.1 × 10 12 viral particles per milliliter (VP/mL) [ 30 ].…”

“…MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; Sigma Chemical Co., St. Louis, MO, USA] assay was used to determine in vitro killing effect of Ad-ΔB/TRAIL+Ad-ΔB/IL-12 on two human HCC cell lines (Hep3B and HuH7) and compared with their effect on the normal human liver cells (WRL68) as previously described [ 29 ]. Briefly, the Hep3B, HuH7 and WRL68 cells were plated onto 24-well plates at about 60 to 70 % confluence, and then treated with PBS, Ad-ΔB, or Ad-ΔB/TRAIL+Ad-ΔB/IL-12 at the indicated MOIs (5 and 10 for Hep3B; 10 and 20 for Huh7; and 20 and 50 MOIs for WRL68).…”

“…Thereafter, the average spectrophotometric readings of the absorbance at 540 nm from three replicates of each treatment were determined in a microplate reader (Bio-Rad 680, Hercules, CA). Cell viability in each well was calculated according the following formula: cell survival = (absorbance value of treated cells-blank)/(absorbance value of untreated control cells), and expressed as the percentage of untreated control cells [ 29 ].…”

“…Survival mice that their imaged signals reached around 2 × 10 8 p/s were enrolled as positive HCC-bearing mice, and were thereafter randomized into three groups ( n = 10/group) and systemically treated with: PBS alone (Group 1), Ad-ΔB (Group 2), and Ad-ΔB/TRAIL + Ad-ΔB/IL-12 (Group 3). The dosage regimen of each OAd was 1 × 10 10 VP (diluted in 200 μL PBS) injected intravenously through the caudal vein and repeated three times every other day [ 29 ]. Three days after administration of the last dose of each treatment strategy, four mice from each group were randomly selected, euthanized under general anesthesia (sodium pentobarbital; 120 mg/kg, i.p.…”

Recent advances in oncolytic adenovirus therapies for cancer

“…An Armed Onc.Ad has been developed expressing FP3 (soluble VEGF receptor 3), which reduces cancer cell VEGF expression and HUVEC proliferation, and has increased oncolytic effect. This Armed Onc.Ad demonstrated enhanced anti-tumor effect in two lung cancer xenograft models [22]. When combined, two breast cancer-selective Onc.Ads engineered to express soluble Flt1 (a portion of VEGF receptor 1) or soluble DII4 (Delta-like 4), which negatively regulate Notch signaling to inhibit maturation of endothelium, provided a significant reduction in vascular formation and doubled animal survival in ER-negative breast cancer xenograft models [23].…”