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. 2020 Mar 13;11:1395. doi: 10.1038/s41467-020-15229-5

Fig. 1. Generation and characterization of an oncolytic vaccinia virus coexpressing a mouse PD-L1 inhibitor and GM-CSF.

Fig. 1

a A schematic diagram of recombinant vaccinia virus (VV) shuttle vectors that express GM-CSF or/and iPDL1 (soluble PD-1-Fc). vTK, VV thymidine kinase gene; R and L, right and left flank sequences; RFP, red fluorescent protein. b Expression and secretion of iPDL1 from infected MC38 tumor cells infected with the indicated VVs. Anti-IgG Fc (Licor 926-32210; upper) or anti-PD-1 (Biolegend 114101; lower) was used for western blot with reducing or non-reducing loading buffer. The experiment was repeated twice. c, d Serum iPDL1 and GM-CSF levels in different VV-treated MC38-bearing mice at 2 days post-virus injection c. Kinetics of iPDL1 levels in injected tumors or sera of the VV-iPDL1/GM-treated mice d. n = 3 independent samples. Data are presented as the means ± SD. The experiment was repeated twice. e Purified iPDL1 binds to PD-L1+ tumor cell. Upper panel: flow cytometric analysis of PD-L1 expression on shPD-L1/MC38 tumor cells that were transduced with PD-L1-shRNA and wild-type MC38 cells. Lower panel: shPD-L1/MC38 cells and wild-type MC38 cells were incubated with 50 μg/mL of purified iPDL1, an irrelevant MAGE3-IgG Fc fusion protein, or IgG control, followed by staining with an anti-IgG Fc for flow cytometry. f Inhibition of PD-1/PD-L1 binding by purified iPDL1 protein using ELISA. An anti-PD-L1 antibody was used as a positive control; n = 3 independent samples. g iPDL1-mediated ADCC. ADCC Reporter Bioassays were performed in triplicate wells, and the concentrations of iPDL1 protein and control IgG Fc used for this assay are indicated; n = 3 independent samples. Data presented as the means ± SD. The experiment was repeated twice. Significant differences are indicated as ***P < 0.001, or ****P < 0.0001 using two-tailed student’s t-test. h CD11c+ DC frequency in monocyte cultures in the presence of culture media of MC38 cells infected with VV-RFP, VV-GM, VV-iPDL1/GM, or GM-CSF as a positive control, and IL-4. i Viral replication in vitro; n = 3 independent samples. j Replication and biodistribution of VV after intratumor injections. Data presented as the means ± SD. The experiment was repeated twice.