Combination Immunotherapy with Vaccine and Oncolytic HSV Virotherapy Is Time Dependent

Abstract

Oncolytic virotherapy or immunovirotherapy is a strategy that utilizes viruses to selectively infect and kill tumor cells while also stimulating an immune response against the tumor. Early clinical trials in both pediatric and adult patients using oncolytic herpes simplex viruses (oHSV) have demonstrated safety and promising efficacy; however, combinatorial strategies designed to enhance oncolysis while also promoting durable T-cell responses for sustaining disease remission are likely required. We hypothesized that combining the direct tumor cell killing and innate immune stimulation by oHSV with a vaccine that promotes T cell-mediated immunity may lead to more durable tumor regression. To this end, we investigated the preclinical efficacy and potential synergy of combining oHSV with a self-assembling nanoparticle vaccine codelivering peptide antigens and Toll-like receptor 7 and 8 agonists (referred to as SNAPvax),which induces robust tumor-specific T-cell immunity. We then assessed how timing of the treatments (i.e., vaccine before or after oHSV) impacts T-cell responses, viral replication, and preclinical efficacy. The sequence of treatments was critical, as survival was significantly enhanced when the SNAPvax vaccine was given prior to oHSV. Increased clinical efficacy was associated with reduced tumor volume and increases in virus replication and tumor antigen-specific CD8+ T cells. These findings substantiate the criticality of combination immunotherapy timing and provide preclinical support for combining SNAPvax with oHSV as a promising treatment approach for both pediatric and adult tumors.

Figure 1.

In vitro and in vivo sensitivity of TC-1 cells to oHSV. (A) TC-1 cells (3000/well) were plated into a 96-well plate. 24 hours later, G207 was added at 0-100 PFU/cell. Cell viability was assessed with alamarBlue 72 hours after addition of virus. The dose required to kill 50% of the cells (LD50) was 4.9 ± 0.8 PFU/cell. Data are mean ± SD. (B) C57BL/6 mice (n= 5-10 per group) implanted with 10⁵ TC-1 tumor cells were administered oHSV or saline intratumorally on day 14. Tumors were measured at the indicated time points. Data represent mean ± SEM. PFU, plaque-forming units. Significance was determined by a multiple unpaired t test.

Figure 2.

SNAPvax E7 combined with oHSV reduced tumor burden and prolonged survival. (A) C57BL/6 mice (n= 5 to 8 per group) implanted with 10⁵ TC-1 tumor cells subcutaneously (SC) in the flank were vaccinated either with SNAPvax E7 (abbreviated “E7”) or an irrelevant/scrambled antigen as a control on day 14 after implantation. G207 or saline was administered intratumorally on day 21. (B) TC-1 tumor growth curves. Data represent mean ± SEM. Significance was determined by two-way ANOVA with Bonferroni correction (**, p < 0.01; ****, p < 0.0001). (C) Survival in mice bearing TC-1 flank tumors. Mice were sacrificed when tumor size reached >1500 mm³ or if a mouse became moribund. Statistics were assessed by log rank test (*, p < 0.05; **, p < 0.01, ****, p < 0.0001).

Figure 3.

SNAPvax E7 prior to oHSV reduced tumor burden and prolonged survival compared to oHSV prior to SNAPvax. (A) C57BL/6 mice (n= 3-7 per group) implanted with 10⁵ TC-1 tumor cells SC in the flank were vaccinated with SNAPvax E7 (abbreviated “E7”) or control on days 14 (E7-oHSV) or 21 (oHSV-E7) after implantation. oHSV or saline was administered intratumorally on day 14 (oHSV-E7) or 21 (E7-oHSV). (B) TC-1 tumor growth curves. Data represent mean ± SEM. Significance was determined by two-way ANOVA with Bonferroni correction (****, p < 0.0001). (C) Survival in mice bearing TC-1 flank tumors. Mice were sacrificed when tumor size reached >1500mm³ or if a mouse became morbid. Statistics were assessed by log rank test (**, p < 0.01). (D) Percent IFN-γ+ of total CD8 positive T cell and (E) percent IFN-γ+ of total CD4 positive T cell from whole blood represented as fold change from naïve on day 24 is shown. Data represent mean ± SEM. Significance was determined by one-way ANOVA with Bonferroni correction (**, p < 0.01).

Figure 4.

SNAPvax prior to oHSV treatment resulted in a directed E7 CD8+ T cell response with enhanced oHSV replication. Tumors treated with either SNAPvax E7 (abbreviated “E7”), oHSV, or vehicle were retreated 7 days later with an alternate therapy (n = 4-5/group). Mice were sacrificed on (A) day 1 or (B) day 3 following the second treatment and percent E7 tetramer+ from CD8+ T cells was calculated. Data represent mean ± SEM. Significance was determined by one-way ANOVA with Bonferroni correction (****, p < 0.001) (C) Tumors treated 7 days prior with E7 or saline were treated with oHSV (1 x 10⁷ PFU) and were harvested at Day 1 and Day 3 after oHSV injection (n = 4 per group). Viral recovery was measured from harvested tumors by limiting dilution plaque assay and normalized by tumor weights (PFU/g). Data are represented as mean ± SEM. Significance was determined using a multiple unpaired t test with the Holm-Šídák multiple comparison test (*, p < 0.05; **, p < 0.01).