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. 2022 Jun 10;10(6):e004762. doi: 10.1136/jitc-2022-004762

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© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See https://creativecommons.org/licenses/by/4.0/.

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Generation of an oncolytic OVH virus expressing a single-chain variable fragment against PD-1. (A) A T cell activation assay was performed with serial dilutions of mouse anti-PD-1 antibodies. Activated human PBMCs were cocultured with 293T/hPD-L1 cells in the presence of increasing mouse anti-PD-1 antibodies for 3 days. The IL-2 levels in the supernatant were assayed by ELISA. (B) The reactivity of anti-PD-1 antibodies (PEM, NIV and hu17D5) against the human PD-1 protein was determined by an indirect CEIA. (C) The blocking activity of anti-PD-1 antibodies was determined by a blocking CEIA. (D) Genetic map showing the coding gene of the hPD-1scFv. The variable regions of the light chain and heavy chain coding genes of hu17D5 were linked by a G₄S sequence, and expression was driven by the viral CMV promoter and a rabbit beta-globin intron. SP: a secretion signal sequence, VK: the variable region sequence of the light chain, VH: the variable region sequence of the heavy chain. (E) The blocking activity of hPD-1scFv and control anti-PD-1 antibodies was determined by a blocking CEIA. (F) The T cell activation activity of hPD-1scFv and control anti-PD-1 antibodies was determined by a T cell activation assay. (G) Schematic of the oncolytic viruses used in this study. Top: genetic map of wild-type HSV-1 (KOS strain). Middle: genetic map of OVH, with deletion of two copies of γ34.5 and ICP0, hTert promoter regulation of ICP27, and insertion of the GFP gene. Bottom: genetic map of YST-OVH showing the inserted coding gene of the hPD-1scFv. (H) hPD-1scFv yields from the supernatants of YST-OVH-infected U-2 OS cells at different time points (MOI=1). (I) Western blot analysis of various proteins in virus-infected cells. (J) Replication of the parental OVH strain and YST-OVH in U-2 OS cells compared with that of wild-type HSV-1. (K) The T cell activating activity of parental hu17D5 and purified hPD-1scFv from virus-infected cell supernatants was determined. (L) Jurkat-Lucia NFAT cells were cocultured with irradiated MDA-MB-231 cells and then stimulated with anti-CD3 and anti-CD28 antibodies in the presence of purified hPD-1scFv. Luciferase activity was measured 24 hours after stimulation. (M) The therapeutic efficacy of hPD-1scFv was evaluated in a syngeneic Hepa1-6 tumor model. Data represent results from one of two (M) independent experiments with n=6 per group. All values are presented as the mean±SEM; repeated-measure ANOVA (M) or unpaired two-tailed Student’s t-test (L). HSV-1, herpes simplex virus type 1. **p<0.01; CEIA, chemiluminescence immunoassay; OVH, oncolytic herpes simplex virus-1; YST-OVH, oncolytic herpes simplex virus-1 expressing PD-1 inhibitors; ANOVA, analysis of variance.