Preclinical Development of Oncolytic Immunovirotherapy for Treatment of HPVPOS Cancers

Abstract

Immunotherapy for HPV^POS malignancies is attractive because well-defined, viral, non-self tumor antigens exist as targets. Several approaches to vaccinate therapeutically against HPV E6 and E7 antigens have been adopted, including viral platforms such as VSV. A major advantage of VSV expressing these antigens is that VSV also acts as an oncolytic virus, leading to direct tumor cell killing and induction of effective anti-E6 and anti-E7 T cell responses. We have also shown that addition of immune adjuvant genes, such as IFNβ, further enhances safety and/or efficacy of VSV-based oncolytic immunovirotherapies. However, multiple designs of the viral vector are possible-with respect to levels of immunogen expression and method of virus attenuation-and optimal designs have not previously been tested head-to-head. Here, we tested three different VSV engineered to express a non-oncogenic HPV16 E7/6 fusion protein for their immunotherapeutic and oncolytic properties. We assessed their profiles of efficacy and toxicity against HPV^POS and HPV^NEG murine tumor models and determined the optimal route of administration. Our data show that VSV is an excellent platform for the oncolytic immunovirotherapy of tumors expressing HPV target antigens, combining a balance of efficacy and safety suitable for evaluation in a first-in-human clinical trial.

Keywords: HPV positive cancer; VSV immunovirotherapy; preclinical.

Figure 1

Characterization of VSV Expressing E7/6 Fusion Protein (A) Genome organization of VSV vectors expressing HPV16 E7/6. (B) E7/6 fusion (top row) and VSV-G (bottom row) protein expression was assessed by western blot. BHK cells were infected with indicated viruses at a MOI of 3. A wild-type E7-expressing TC-1 cell line was used as a positive control (top row). Viral growth curves on (C) BHK-21 and (D) TC-1 cells infected at a MOI of 0.002 and 0.1, respectively. In vitro cytotoxic activity of VSV in (E) TC-1 and (F) MPC-11 murine tumor cell lines. An MTS cell-viability assay following infections with increasing MOIs were performed at 48 hr after infections. Error bars, SD of the mean.

Figure 2

Efficacy of VSV-E7/6 Vectors in the Murine TC-1 Tumor Model Kaplan-Meier survival curve of C57BL/6 mice (n = 10) bearing TC-1 tumors following three virus doses, injected either i.v. (5 × 10⁸ TCID₅₀) or i.t. (4.2 × 10⁸) every other day (A and B, respectively) starting 7 days after tumor implantation. Kaplan-Meier survival curves were compared by log rank (Mantel-Cox) analysis. A value of p < 0.05 was considered statistically significant. (C) HPV16 E7/6 specific T cell response detected by ELISPOT assay in surviving mice (VSV-M51R-E7/6 group), following restimulation of splenocytes with either medium alone or a pool of overlapping E7 and E6 mixpeptides. Error bars, SD of the mean.

Figure 3

Efficacy and Toxicity of VSV-E7/6 Vectors in the Murine MPC-11 Tumor Model Tumor volume measurements of BALB/c mice (n = 10) bearing MPC-11 tumors after treatment with either a single i.v. (A) or i.t. (B) injection of virus (5 × 10⁶ TCID₅₀) on day 7 after tumor implantation. Kaplan-Meier survival curve of the same groups of mice after i.v. (C) or i.t. (D) viral administration. A value of p < 0.05 was considered statistically significant.

Figure 4

Profiles of Blood and Liver Function Enzymes in MPC-11 Tumor-Bearing Mice after Treatment with VSV-E7/6 Vectors (A) At the time of euthanasia in saline or treatment groups, blood was collected by retro orbital bleed to monitor changes in blood composition; WBC, total white blood cell; LYM, lymphocyte; MON, monocyte; NEU, neutrophil; HGB, hemoglobin; PLT, platelet. The dot lines indicate the 95% interval of each parameter measure in 158 normal 8- to 10-week-old female BALB/c mice. (B) Profiles of liver function enzymes at the time of euthanasia in saline or treatment groups. Blood was collected by retro orbital bleed to monitor liver function; BUN, blood urea nitrogen; CRE, creatinine; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase. The dotted lines indicate the 95% interval of each parameter measured from 121–133 normal 8- to 10-week-old female BALB/c mice. Error bars, SD of the mean.

Figure 5

In Vivo and In Vitro Efficacy and Safety following Multiple Administrations of VSV-M51R-E7/6 and In Vitro Cytotoxic Effect on Human Cells (A) Kaplan-Meier survival curve of BALB/c mice (n = 10) bearing MPC-11 tumors after treatment with three doses of VSV-M51R-E7/6 (5 × 10⁸ TCID₅₀/dose), injected either i.t. or i.v. every other day, starting 7 days after tumor implantation. p < 0.0001 (saline versus i.v. injection); p = 0.0021 (saline versus i.t. injection) Kaplan-Meier survival curves were compared by log rank (Mantel-Cox) analysis. A value of p < 0.05 was considered statistically significant. (B) In vitro cytotoxic activity of VSV-M51R-E7/6 in a panel of human cell lines. The indicated cell monolayers were infected with VSV-M51R-E7/6 (solid line) or VSV-GFP (dotted line) at the indicated MOIs. An MTS cell viability assay was performed 72 hr post-infection. HEKa, normal primary epidermal keratinocytes; HT1080, fibrosarcoma; Mel624, melanoma; and LoVo, colorectal adenocarcinoma. Error bars, SD of the mean.

Figure 6

Optimizing Route of Administration and Dosing Schedule of VSV-M51R-E7/6 Therapy (A) Tumor volumes of C57BL/6 (n = 10) mice bearing TC-1 tumors after treatment with either four i.m. (left) or i.v. (right) VSV-M51R-E7/6 (5 × 10⁸ TCID₅₀/injection) injections every other day. (B) Corresponding Kaplan-Meier survival curve of mice treated with VSV-M51R-E7/6 by i.m. or i.v. injections. (C) Kaplan-Meier survival curves of mice treated with either one or four i.v. injections of VSV-M51R-E7/6 (5 × 10⁸ TCID₅₀/injection). (D) Kaplan-Meier survival curve of mice after treatment with either a single i.v. or i.t. injection or following concomitant i.v. and i.t. injection with VSV-M51R-E7/6 (5 × 10⁸ TCID₅₀ total dose in each case, splitting the dose in half for concomitant i.v. and i.t. administration) starting 7 days after tumor implantation. An additional i.t. dose (5 × 10⁸ TCID₅₀) was given to all groups 10 days after the first injection. Kaplan-Meier survival curves were compared by log rank (Mantel-Cox) analysis. A value of p < 0.05 was considered statistically significant.

Figure 7

Effect of VSV-M51R-E7/6 Administration on the Tumor Microenviroment and Generation of Antigen-Specific T Cell Immune Responses (A) TC-1 tumors (n = 5) were harvested 7 days after viral therapy and analyzed by flow cytometry. (B) Mouse IFNγ ELISPOT assay was performed on splenocytes (n = 5) harvested 20 days after viral administration. (C) Tumor volume measurements at day 20 after i.t., i.v., or concomitant i.v. + i.t. administration of VSV-M51R-E7/6 (5 × 10⁸ TCID₅₀ total dose, splitting the dose in half for concomitant i.v. + i.t. injection) starting 7 days after tumor implantation. The data shown are the average of experiment with five mice per group. p values were calculated using non-parametric Mann-Whitney t test; *p < 0.05, **p < 0.01.

Figure 8

VSV-M51R-E7/6 Efficacy in the TC-1 Tumor Model Is Dependent on the Generation of a CD8⁺ T Cell Response (A) Tumor volume measurements and (B) Kaplan-Meier survival curves of VSV-M51R-E7/6 treated C57BL/6 (n = 10) mice bearing TC-1 tumors after depletion of different subsets of immune cells. Mice were treated intraperitoneally with depleting antibodies (200 μg/mouse) starting 4 days after tumor implantation and twice weekly thereafter (100 μg/mouse). Mice received a concomitant i.v. and i.t. injection of VSV-M51R-E7/6 (5 × 10⁸ TCID₅₀ total dose, splitting the dose in half for concomitant i.v. and i.t. injection) or saline, starting 7 days after tumor implantation. Kaplan-Meier survival curves were compared by log rank (Mantel-Cox) analysis. A value of p < 0.05 was considered statistically significant.