Fig. 1: Construction and characterization of OV-Q1, OV-αCD47-G1, and OV-αCD47-G4. | Nature Communications

Fig. 1: Construction and characterization of OV-Q1, OV-αCD47-G1, and OV-αCD47-G4.

From: An oncolytic virus expressing a full-length antibody enhances antitumor innate immune response to glioblastoma

Fig. 1

a Flow cytometric assay of CD47 expression on GBM patient-derived GBM30, GBM43, and BT422 cells. b Human CD47 binding affinity of αCD47-G1 and αCD47-G4 purified from CHO cells as assessed by flow cytometry. U251T2 human GBM cells were incubated with an increasing concentration of αCD47-G1 or αCD47-G4 mAbs followed by staining with an APC-conjugated anti-human Fc. c Inhibition of anti-CD47 mAb binding by αCD47-G1 and αCD47-G4 mAbs. U251T2 cells were incubated with an increasing concentration of αCD47-G1 or αCD47-G4 mAb followed by incubation with a conjugated anti-human CD47 antibody and assessed by flow cytometry. d Schematic of oncolytic viruses used in this study. Top: genetic map of wild-type HSV-1. Second: genetic map of control oHSV, OV-Q1, with deletion of two copies of γ34.5, dysfunction of ICP6, and insertion of the GFP gene. Third: genetic map of OV-αCD47-G1 showing the inserted coding gene of the IgG1 version of anti-CD47 (αCD47-G1). Fourth: genetic map of OV-αCD47-G4 showing the inserted coding gene of the IgG4 version of anti-CD47 (αCD47-G4). The light-chain and heavy-chain coding genes of αCD47-G1 and αCD47-G4 are linked by a T2A sequence and are driven by the viral pIE4/5 promoter. LC light chain, HC heavy chain, hIgκ human Igκ light chain, hIgG1 human IgG1 heavy chain, hIgG4 human IgG4 heavy chain. e Immunoblotting performed with concentrated supernatants of engineered CHO cells and oHSV-infected U251T2 human GBM cells by anti-human Fc or anti-human Igκ antibody. f αCD47-G1 and αCD47-G4 yields from supernatants of OV-αCD47-G1- and OV-αCD47-G4-infected U251T2 cells as determined by ELISA assay. g U251T2 human GBM cells were infected with OV-Q1, OV-αCD47-G1, or OV-αCD47-G4 at the indicated MOIs. Cell lysis was analyzed at 3 days after infection by CCK8 cell viability assay. h U251T2 cells were infected with OV-Q1, OV-αCD47-G1, or OV-αCD47-G4 at an MOI of 2. The supernatants were harvested at indicated time points for viral reproduction using a plaque assay in Vero cells. Experiments in (a−c) and (e−h) are representative of three independent experiments with similar data. Data are presented as mean values ± SD (n = 3).

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