Local Delivery of OncoVEX^mGM-CSF Generates Systemic Antitumor Immune Responses Enhanced by Cytotoxic T-Lymphocyte–Associated Protein Blockade

OncoVEX^mGM-CSF-induced lysis and HSV-1 virus are only detected in OncoVEX^mGM-CSF-injected tumors. A–D, Growth of OncoVEX^mGM-CSF (dose response 3 × 10⁴ to 3 × 10⁶ PFU) and vehicle-treated A20 tumors on the injected and contralateral side. Each line represents the growth pattern of a single tumor (n = 10/group). Data are representative of at least three independent experiments. E, Kaplan–Meier analysis of median survival of mice treated with OncoVEX^mGM-CSF vs. vehicle. Events were recorded when tumor volume exceeded 800 mm³. F, ddPCR quantification analysis of viral HSV-1 DNA. Bars or points represent the mean copy-number variation (CNV) and SD for each dose or time point (n = 4). Dose–response analysis was performed at 24 hours. Time response analysis was performed at 5 × 10⁶ PFU. G, Relative expression of viral gene products (ICP27 and thymidine kinase) is shown for injected and contralateral tumors as well as inguinal TDLNs draining each. Time points as indicated. Viral gene product expression is representative of two independent experiments with n = 5/group (*, P < 0.001). H, Sections from vehicle- and OncoVEX^mGM-CSF-injected and contralateral A20 tumors at 96 hours postinjection were stained by H&E or an antibody-specific for an HSV-1 antigen. IHC data are representative of three independent experiments, n = 5/group. I, In vivo detection of OncoVEX^mGM-CSF HSV-1 thymidine kinase activity by PET/CT imaging was performed using the radiolabeled penciclovir analog [¹⁸F]FHBG. [¹⁸F]FHBG accumulation in OncoVEX^mGM-CSF-injected tumors compared to contralateral or vehicle-injected tumors.

Treatment with OncoVEX^mGM-CSF induces a localized inflammatory response. A, Bilateral A20 tumor sections from OncoVEX^mGM-CSF (single dose of 5 × 10⁶ PFU) and vehicle control mice collected at time points indicated were processed to extract RNA. Indicated type I IFN gene signature was measured as the geometric mean of five different IFN inducible genes using Fluidigm qPCR. Statistics shown are for OncoVEX^mGM-CSF-injected tumors vs. vehicle-injected using a one-way ANOVA (Dunnett's multiple comparison test). B, Expression of the GM-CSF, IFNγ, CXCL2, and CXCL10 was measured in vehicle and OncoVEX^mGM-CSF-treated A20 tumor between 1 and 96 hours. Data are representative of two independent experiments. C, Viable cell counts obtained from LN disaggregates. Viable cell count by trypan exclusion harvested 96 hours after treatment and flow cytometric analysis of CD69⁺ CD3⁺ lymphocytes from vehicle- and OncoVEX^mGM-CSF-treated mice at 2, 5, and 10 days after treatment. Data are representative of two independent experiments n = 10/group; statistics shown are for one-way ANOVA with Sidak correction for multiple testing, with comparators preselected by matched time point (OncoVEX^mGM-CSF vs. vehicle) (*, P < 0.0001; **, P < 0.01; ***, P < 0.05).

OncoVEX^mGM-CSF increases immune cells in both injected and contralateral tumor. A, Relative expression of CD3E, CD8A, and CD103 (ITGAE) was analyzed from vehicle-injected and OncoVEX^mGM-CSF-injected and corresponding contralateral tumors. Gene expression analysis detects increased infiltration into both OncoVEX^mGM-CSF-injected and contralateral tumors at 168-hour time points, compared with vehicle-injected tumors. Gene expression analysis is representative of two independent experiments with n = 5/group. B and C, Morphometric analysis of A20 tumor serial sections by IHC. A20 tumor sections from OncoVEX^mGM-CSF (single dose of 5 × 10⁶ PFU) and vehicle control mice 168 hours (or as indicated in Fig. 4C) after injection were subjected to morphometric analysis, and the percent area of ROI was calculated for CD3, CD8, and CD103. Statistics were generated using unpaired Mann–Whitney nonparametric test. D and E, FACS analysis of A20 tumors following OncoVEX^mGM-CSF (single dose of 5 × 10⁶ PFU) and vehicle treatment at the indicated time point (D) and at 96 hours (E). Data are representative of three independent experiments (*, P < 0.0001; **, P < 0.01; ***, P < 0.05).

T cells from OncoVEX^mGM-CSF-treated tumor-bearing mice specifically and functionally recognize tumor antigens. A, Ex vivo restimulation reveals systemic antitumor reactivity. Splenic T cells were isolated from A20 tumor-bearing animals on days 4, 7, 11, and 14 following OncoVEX^mGM-CSF (5 × 10⁶ PFU) intratumoral injection and rechallenged ex vivo for 24 hours with either CT-26 or A20 tumor cells. Data are depicted as number of IFNγ-positive spots per 100,000 plated T cells. B–E, Efficacy of OncoVEX^mGM-CSF following depletion of CD8⁺ T cells. Each line represents the growth pattern of a single A20 tumor (n = 10/group). Mice were treated with anti-CD8 antibody or control isotype. Following confirmation of CD8⁺ T-cell depletion, the mice where inoculated (contralaterally) with A20 cells, randomized into four groups when tumors reached 160 mm³, and treated with vehicle or OncoVEX^mGM-CSF at 5 × 10⁶ PFU every 3 days for the first week. Tumor volume and body weights were measured with calipers or an analytical scale, respectively. F, Kaplan–Meier analysis of median survival of mice treated with OncoVEX^mGM-CSF vs. vehicle, with or without anti-CD8. Events were recorded when tumor volume exceeded 800 mm³. G, Schematic of adoptive cell transfer model. H–J, Immune cells from naïve BALB/C mice, BALB/C mice bearing A20 tumors or BALB/C mice cured of their A20 tumors with OncoVEX^mGM-CSF treatment were collected and transplanted into recipient BALB/C mice. Mice in all groups were then challenged with A20 cells. Each line represents an individual A20 tumor followed using calipers for 50 days. All data are representative of two independent experiments (*, P < 0.01; **, P < 0.0001).