Figure 4.

Co-infection of Onc.Ad with HDAd Expressing Both IL-12p70 and PD-L1 Mini-Antibody Amplifies IL-12p70 and PD-L1-Blocking Antibody Expression with Oncolysis In Vitro

(A) Schematic structure of HDAd encoding human IL-12p70 and anti-PD-L1 mini-antibody expression cassettes (HDAdIL12_PDL1). A549 cells were infected with 100 vps/cell of HDAdEGFP, HDAdIL-12, HDAdPD-L1, or HDAdIL12_PDL1. Media were collected 48 hr post-infection. Medium samples were subjected to IL-12p70 ELISA and western blotting for PD-L1 mini-antibody, which was detected by anti-HA antibody. (B) FaDu and SCC-47 cells were infected with a total of 10 vps/cell of HDAdIL12_PDL1 or Onc.Ad or with CAd12_PDL1 (Onc.Ad:HDAd, 1:10). Medium samples were collected 48 hr post-infection. The levels of IL-12p70 and PD-L1 mini-antibody in medium samples were quantified by IL-12p70 ELISA assay and western blotting for PD-L1 mini-antibody, respectively. Data are presented as means ± SD (n = 4). *p < 0.001. (C) DNA samples were extracted 48 hr post-infection, and Onc.Ad and HDAd vector copy numbers were measured by qPCR. Data were normalized with human genomic GAPDH. Data are presented as means ± SD (n = 4). *p = 0.008. CAdVEC: Onc.Ad:HDAdIL12_PDL1, 1:10. (D) FaDu and SCC-47 cells were infected with increasing doses of HDAdIL12_PDL1 or Onc.Ad or with CAdVEC (Onc.Ad: HDAdIL12_PDL1, 1:10). Viable cells were analyzed at 96 hr by MTS assay. Data are presented as means ± SD (n = 6).