Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT)

Abstract

Background: Oncolytic virotherapy with vaccinia virus (VV) can lead to effective anti-tumor immunity by turning "cold" tumors into "hot" tumors. However, its therapeutic potential is affected by the tumor's local immunosuppressive tumor microenvironment (TME). Therefore, it is necessary to explore the use of immune checkpoint inhibitors to arm oncolytic VVs to enhance their anti-tumor efficacy.

Methods: A novel recombinant oncolytic VV, VV-α-TIGIT, which encoded a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT) was generated by homologous recombination with a shuttle plasmid. The anti-tumor efficacy of the VV-α-TIGIT was investigated in several subcutaneous and ascites tumor models.

Findings: The functional α-TIGIT was sufficiently produced and secreted by tumor cells infected with VV-α-TIGIT, which effectively replicated in tumor cells leading to significant oncolysis. Intratumoral injection of VV-α-TIGIT improved anti-tumor efficacy in several murine subcutaneous tumor models compared to VV-Control (without α-TIGIT insertion). Intraperitoneal injection of VV-α-TIGIT achieved approximately 70% of complete tumor regression in an ascites tumor model. At the same time, treatment with VV-α-TIGIT significantly increased the recruitment and activation of T cells in TME. Moreover, the in vivo anti-tumor activity of VV-α-TIGIT was largely dependent on CD8⁺ T cell-mediated immunity. Finally, the tumor-bearing mice cured of VV-α-TIGIT treatment resisted rechallenge with the same tumor cells, suggesting a long-term persistence of tumor-specific immunological memory.

Interpretation: The recombinant oncolytic virus VV-α-TIGIT successfully combines the advantages of oncolytic virotherapy and intratumorally expression of immune checkpoint inhibitor against TIGIT. This novel strategy can provide information on the optimal design of novel antibody-armed oncolytic viruses for cancer immunotherapy.

Funding: This work was supported by the National Natural Science Foundation of China (81773255, 81472820, and 81700037), the Science and Technology Innovation Foundation of Nanjing University (14913414), and the Natural Science Foundation of Jiangsu Province of China (BK20171098).

Keywords: Cancer immunotherapy; Checkpoint inhibitors; Oncolytic virus; TIGIT; Vaccinia virus.

Fig. 1

Generation and characterization of recombinant oncolytic VVs. (A) A schematic diagram of the recombinant VVs with (VV-α-TIGIT) or without (VV-Control) α-TIGIT gene. TK-R, right flank sequences of thymidine kinase gene; TK-L, left flank sequences of thymidine kinase gene; GPT, guanine phosphoribosyl transferase; EGFP, enhanced green fluorescent protein; p-7.5k, vaccinia virus p-7.5k early/late promoter; p-se/l, synthesized vaccinia virus early/later promoter; T2A, thoseaasigna virus 2A; E2A, equine rhinitis A virus 2A; (B) Expression and secretion of α-TIGIT in VV-infected Hela-S3 cells. Hela-S3 cells were infected with indicated VVs at MOI of 1 for 24 h, and the levels of α-TIGIT antibody in the cell lysates and supernatants were detected by a luciferase assay. Data are expressed as means ± SD. The experiment was repeated three times. Statistical differences were estimated using the student's t-test. ^⁎⁎⁎⁎P < 0.0001. (C) Secretion of α-TIGIT in VV-infected 4T1, CT26, MC38, and H22 cells. The detection of α-TIGIT is similar to that described in (B). Statistical differences were estimated using the ANOVA. ^⁎⁎⁎⁎P < 0.0001. (D) Luciferase-linked immunosorbent assay was used to investigate the binding of the secreted α-TIGIT antibody to the recombinant TIGIT (r-TIGIT) protein. The 96-well plate was coated with r-TIGIT (10 μg/ml), and the supernatants of cells infected with VVs were added to the plate. After three rounds of washing, a luciferase assay was used to verify the binding of the secreted α-TIGIT antibody to the r-TIGIT protein. Data are expressed as means ± SD. The experiment was repeated three times. Statistical differences were estimated using analysis of variance (ANOVA). ^⁎⁎⁎⁎P < 0.0001.

Fig. 2

Oncolysis and replication of VVs in tumor cells. (A) 4T1, CT26, MC38, and H22 cells were plated into 96-well plates and infected with VVs at the indicated MOI for 48 and 72 h. The cell viability was determined by MTT assay for 4T1, CT26, and MC38 cells and was determined by CCK8 assay for H22 cells. Data represent the mean ± standard deviation (SD) of three independent experiments. (B) 4T1, CT26, and MC38 cells were plated into a 96-well plate and infected with VVs at the indicated MOI for 72 h, and oncolytic ability was determined by crystal violet staining. The figure represents one of the three experiments performed. (C) 4T1, CT26, MC38, and H22 cells were infected with VVs at MOI of 0.1, cells were harvested at designated time points and progeny viral particles were quantified by titration assays (TCID50).

Fig. 3

Expression of PVR/CD155 and PD-L1 on tumor cells. Flow cytometry histogram representative of the expression of PVR/CD155 (A) and PD-L1 (B) on 4T1, CT26, MC38 and H22 cells. To analyze PVR/CD155, PE-conjugated rat-anti-mouse IgG2a was used as an isotype control. To analyze PD-L1, PE-conjugated rat-anti-mouse IgG2b was used as an isotype control. Data are presented as the mean fluorescence intensity (MFI) ± SD of three independent assay. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4

VV-α-TIGIT has enhanced antitumor activity in a breast cancer model. (A) The mice treatment scheme. The breast cancer subcutaneous (S.C.) model was established by implantation of 2 × 10⁵ 4T1 cells on the right flank of BALB/c mice. When the tumor volume reached approximately 50 mm³ (7 days post tumor inoculation), the tumor-bearing mice were treated with PBS, 1 × 10⁷ PFU of VV-Control, or VV-α-TIGIT at a 2-day interval for 3 times via intratumoral (I.T.) injection. (B) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 15 mice for all groups. (C) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (D) The body weight of the mice. Data are presented as the mean ± SD of 15 mice for all groups. (E) The mice treatment scheme of monotherapy with VV-α-TIGIT or combination therapy with VV-Control and α-TIGIT. The tumor model was established similar to (A). In the combination treatment group, an additional α-TIGIT intraperitoneally (I.P.) treatment was performed during the first intratumoral injection of VV-Control. (F) Tumor volume of mice treated with VV-α-TIGIT or VV-Control plus α-TIGIT was determined every two days. Data are presented as the mean ± SD of 8 mice for each group. (G) Kaplan–Meier survival curves of tumor-bearing mice treated with VV-α-TIGIT or VV-Control plus α-TIGIT. (H) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for each group. Statistical differences in tumor volume and body weight among the groups were evaluated using ANOVA. Statistical differences in survival among the groups were evaluated using the Log-Rank test. ns, no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5

Anti-tumor activity of the recombinant VV on colon cancer models. (A) Treatment scheme of MC38 tumor model. The subcutaneous tumor model was established by inoculation of 2 × 10⁶ MC38 cells on the right flank of C57BL/6 mice. Mice were administered intratumorally at the indicated day for 3 times with PBS, 1 × 10⁷ PFU of VV-Control or VV-α-TIGIT. (B) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (C) Kaplan-Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (D) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (E) Treatment scheme of CT26 tumor model. The subcutaneous tumor model was established by inoculation of 5 × 10⁵ CT26 cells on the right flank of BALB/c mice. Mice treatment were similar to (A). (F) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (G) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (H) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (I, J) Immunohistochemistry (IHC) detection of the infiltration of CD8⁺ T cells (I) and microvascular density (MVD) (J) of the tumor in the CT26 subcutaneous tumor model. The tumor model was established as previously described. When the tumor reached approximately 50 mm³, mice were treated i.t. with PBS, 1 × 10⁷ PFU of VV-Control or VV-α-TIGIT. Seven days after VV injection, tumors were collected from mice, and CD8 and CD31 expression were detected by IHC. (K, L) The α-TIGIT levels in tumor and blood. Samples of the tumor lysates and sera were prepared as previously described in materials and methods and a luciferase assay was used to detect the levels of secreted α-TIGIT. (M) Viral titers in tumor and blood. Samples were collected similar to (K, L), and viral titers were quantified by a TCID50 method. Statistical differences in tumor volume, body weight, CD8⁺ T cells, MVD, luciferase activity, and viral titer among the groups were evaluated using ANOVA. Statistical differences in survival among the groups were evaluated using the Log-Rank test. ns, no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6

VV-α-TIGIT enhanced immune-cell infiltration and activation in the tumor microenvironment. (A) The treatment scheme. The tumor model was established by intraperitoneally (I.P.) inoculation of 5×10⁶ H22 cells in C57BL/6 mice. After ascites were formed, the tumor-bearing mice were treated with PBS, 1 × 10⁷ PFU of VV-Control or VV-α-TIGIT at a 2-day interval 3 times via I.P. injection. (B) Dynamic detection of the proportion of the tumor cells and lymphocytes in the ascites by flow cytometry. (C, D) Flow cytometric analysis of the proportion of the tumor cells, lymphocytes, and their subsets in the ascites at day 4 after the first VV-α-TIGIT treatment. (E) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, or VV-α-TIGIT (n=14). (F) The α-TIGIT levels in ascites. Ascites were harvested on day 4 after the first VV-α-TIGIT treatment and a luciferase assay was used to detect the levels of secreted α-TIGIT in ascites. (G) The concentration of murine IFN-γ was detected by an ELISA assay in the ascites of mice on day 4 after the first VV-α-TIGIT treatment. Statistical differences in cell proportion, luciferase activity, and IFN-γ concentration in the ascites were evaluated using ANOVA. Statistical differences in survival of mice among the groups were evaluated using the Log-Rank test. ns, no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 7

The in vivo anti-tumor activity of VV-α-TIGIT depends on CD8⁺ T cells. (A) Schematic diagram of the experiment for CD8⁺ T or NK cell depletion. Ascites tumor model was established as previously described. Three days post tumor inoculation, mice were injected intraperitoneally (I.P.) with 500 ug of anti-mouse CD8α or anti-mouse NK1.1 mAB per mouse. (B) The depletion of CD8⁺ T and NK cells was confirmed by detecting the corresponding cells in ascites by flow cytometry 5 days post the mAB injection. (C) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-α-TIGIT, VV-α-TIGIT plus anti-CD8α, or VV-α-TIGIT plus anti-NK1.1. (D) Flow cytometric analysis of the proportion of the tumor cells, lymphocytes, and their subsets in the ascites. (E) The level of α-TIGIT in ascites was detected by a luciferase assay on day 4 after the first VV-α-TIGIT treatment. (F) The concentration of murine IFN-γ was detected by an ELISA assay in the ascites of mice on day 4 after the first VV-α-TIGIT treatment. Statistical differences in cell proportion, luciferase activity, and IFN-γ concentration in the ascites were evaluated using ANOVA. Statistical differences in survival of mice among the groups were evaluated using the Log-Rank test. ns, no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 8

Treatment of mice with VV-α-TIGIT established long-term tumor-specific immunological memory. (A) Schematic diagram of tumor rechallenge. Ascites tumor model was established and treated as previously described. Mice cured of H22 by VV-α-TIGIT were twice intraperitoneally and once subcutaneously inoculated with the H22 cells, and once subcutaneously inoculated with MC38 cells at the indicated time. (B) Kaplan-Meier survival curves of tumor-naïve or cured mice twice intraperitoneally rechallenged with H22 cells (n=10). (C) Tumor volumes and Kaplan–Meier survival curves of tumor-naïve or cured mice subcutaneously rechallenged with H22 cells (n=10). (D) Tumor volumes and Kaplan–Meier survival curves of tumor-naïve or cured mice subcutaneously rechallenged with MC38 cells (n=10). Statistical differences in tumor volume were evaluated using ANOVA. Statistical differences in survival were evaluated using the Log-Rank test. ns, no significant differences; ****P < 0.0001.