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. 2020 Nov 17:20:12-22.
doi: 10.1016/j.omto.2020.11.001. eCollection 2021 Mar 26.

Recombinant oncolytic adenovirus expressing a soluble PVR elicits long-term antitumor immune surveillance

Hailin Zhang  1 , Yonghui Zhang  1 , Jie Dong  1 , Binghua Li  1 , Chun Xu  1 , Min Wei  1 , Junhua Wu  1   2 , Jiwu Wei  1
Affiliations

Recombinant oncolytic adenovirus expressing a soluble PVR elicits long-term antitumor immune surveillance

Hailin Zhang et al. Mol Ther Oncolytics. .

Abstract

Oncolytic virotherapy (OVT) has been suggested to be effective. However, the suppressive effects of checkpoints and insufficient costimulatory signals limit OVT-induced antitumor immune responses. In this study, we constructed a replicative adenovirus, Ad5sPVR, that expresses the soluble extracellular domain of poliovirus receptor (sPVR). We showed that sPVR can bind to both T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) and CD226, and the binding affinity of sPVR to TIGIT is stronger than that of PVR to CD226. In the H22 hepatocellular carcinoma (HCC) ascites model, Ad5sPVR treatment increased the infiltration of CD8+ T cells and the release of interferon (IFN)-γ, exhibiting an antitumor effect with long-term tumor-specific immune surveillance. In line with this, Ad5sPVR also effectively improved antitumor outcomes in solid tumors. In conclusion, while Ad5sPVR plays a role in oncolysis and transforms cold tumors into hot tumors, sPVR expressed by Ad5sPVR can block the PVR/TIGIT checkpoint and activate CD226, thereby greatly improving the efficacy of OVT. This study provides a new way to develop potential oncolytic viral drugs.

Keywords: CD226; PVR/CD155; T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain; TIGIT; cancer therapy; immune checkpoints; oncolytic virus.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Generation of a novel recombinant adenovirus, Ad5sPVR, expressing the soluble extracellular domain of PVR (sPVR) (A) Recombinant adenovirus constructs. EXO, extracellular domain; Ad5sPVR, recombinant adenovirus encoding sPVR. (B) H22 cells and B16/F10 cells were infected with the recombinant adenovirus (multiplicity of infection [MOI] of 20 and 10, respectively) for 72 h, and the levels of sPVR in the supernatant were determined by western blotting. The molecular weights were estimated using a molecular weight marker. (C) 293T cells were infected with Ad5sPVR, and soluble PVR in the cell culture supernatant was harvested and purified by a nickel column. The affinity of sPVR/TIGIT and sPVR/CD226 was detected using an Octet RED96 protein interaction analysis system.
Figure 2
Figure 2
Replication and oncolytic activities of the recombinant adenovirus Ad5sPVR (A) Hepa1-6, H22, LM3, B16/F10, 4T1, and LLC1 cells were infected with recombinant adenoviruses at an MOI of 5, 20, 2, 10, 5, and 10 and were harvested at various time points. DNA was extracted and the viral copy number was determined by RT-PCR. (B) Hepa1-6, H22, LM3, B16/F10, 4T1, and LLC1 cells were infected with recombinant adenovirus at the indicated MOIs for 72 h, and cell viability was determined by the MTT assay. Virus replication and oncolysis are presented using one-way ANOVA with repeated measures (SPSS). The data are shown as the means ± SD. ns, not significant.
Figure 3
Figure 3
Effect of recombinant adenovirus Ad5sPVR on antitumor immune activation and lymphocyte infiltration (A) Schematic diagram of the experimental setup for adenovirus therapy in H22-challenged mice. On day 0, male C57BL/6 mice were injected intraperitoneally with 5 × 106 H22 cells, and the mice were randomly divided into three groups: saline group, Ad5con group, and Ad5sPVR group. On days 8, 12, and 16, mice in the saline group, Ad5con group, and Ad5sPVR group were injected intraperitoneally with saline, the control virus Ad5con (5 × 108 PFU), and Ad5sPVR (5 × 108 PFU), respectively. On days 12 and 16, before intraperitoneal injection of saline or virus, ascites were collected. (B) Virus replication was measured in the ascites of mice infected with recombinant adenovirus. On day 16, before intraperitoneal injection of saline or virus, ascites were collected and the viral copy number was determined by RT-PCR. (C) On day 16, before intraperitoneal injection of saline or virus, ascites were collected and the concentration of sPVR in ascites was determined by ELISA. (D) On day 16, before intraperitoneal injection of saline or virus, ascites were collected, and CD4+ T cell, CD8+ T cell, and NK cell frequencies in ascites were determined by flow cytometry. (E) On day 16, before intraperitoneal injection of saline or virus, ascites were collected, and immune activity in the TME was detected using a mouse IFN-γ ELISpot assay, and the number of IFN-γ spots in each group was calculated. The data are shown as the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. i.p., intraperitoneal; s.c., subcutaneous; i.t., intratumoral.
Figure 4
Figure 4
Effect of recombinant adenovirus Ad5sPVR on the antitumor immune response and cure rate in the H22 ascites model (A) Schematic diagram of the experimental setup for adenovirus therapy. On day 0, male C57BL/6 mice were injected intraperitoneally with 5 × 106 H22 cells, and the mice were randomly divided into three groups: saline group, Ad5con group, and Ad5sPVR group. On days 8, 12, and 16, mice in the saline group, Ad5con group, and Ad5sPVR group were injected intraperitoneally with saline, the control virus Ad5con (5 × 108 PFU), and Ad5sPVR (5 × 108 PFU), respectively. On days 12 and 16, before intraperitoneal injection of saline or virus, ascites were collected for further study. (B) Virus replication in the ascites of mice infected with recombinant adenovirus. On day 16, before intraperitoneal injection of saline or virus, ascites were collected and the viral copy number was determined by RT-PCR. (C) On day 16, before intraperitoneal injection of saline or virus, ascites were collected and the concentration of sPVR in ascites was determined by ELISA. (D) Survival curves for mice treated with recombinant adenovirus. (E) Mice that had been cured were challenged with 5 × 106 H22 cells on day 90 and day 150 post-inoculation. Naive mice were used as a control. The day of challenge was regarded as day 0 in survival curves. The data are shown as the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 5
Figure 5
Effect of recombinant adenovirus Ad5sPVR on the antitumor immune response in B16/F10 solid tumor models (A) Schematic diagram of the experimental setup for adenovirus therapy in B16/F10-challenged mice. On day 0, male C57BL/6 mice were injected subcutaneously with 2 × 106 B16/F10 cells in the right flank, and the mice were randomly divided into three groups: saline group, Ad5con group, and Ad5sPVR group. On days 8, 9, and 10, mice in the saline group, Ad5con group, and Ad5sPVR group were injected intratumorally with saline, the control virus Ad5con (5 × 108 PFU), and Ad5sPVR (5 × 108 PFU), respectively. On day 13, tumors were dissected. Single-cell suspensions were obtained from the tumor tissue, and cells were counted after trypan blue staining using CountStar. (B) Immune activity in the TME was detected using a mouse IFN-γ ELISpot assay, and the number of IFN-γ spots in each group was calculated. (C) Schematic diagram of the experimental setup for adenovirus therapy. Male C57BL/6 mice received a subcutaneous injection of 2 × 106 B16/F10 cells in the right flank. When the tumor size reached approximately 4–6 mm in diameter, the mice were randomly divided into three groups. On days 8, 9, 10, and 11, mice in the saline group, Ad5con group, and Ad5sPVR group were injected intratumorally with saline, the control virus Ad5con (5 × 108 PFU), and Ad5sPVR (5 × 108 PFU), respectively. (D and E) Tumor volumes were measured using a caliper, and body weight was monitored every day during treatment. (F) Volume of each tumor in the group of mice treated with saline, Ad5con, and Ad5sPVR. The data are shown as the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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