Preclinical safety and activity of recombinant VSV-IFN-β in an immunocompetent model of squamous cell carcinoma of the head and neck

Abstract

Background: Recombinant vesicular stomatitis virus expressing interferon-β (VSV-IFN-β) has demonstrated antitumor activity in vitro and in vivo. In preparation for clinical testing in human squamous cell carcinoma (SCC) of the head and neck, we conducted preclinical studies of VSV-IFN-β in syngeneic SCC models.

Methods: In vitro, VSV-IFN-β (expressing rat or mouse interferon [IFN]-β)-induced cytotoxicity and propagated in rat (FAT-7) or mouse (SCC-VII) SCC cells during normoxia and hypoxia. In vivo, intratumoral administration of VSV-rat-IFN-β or VSV-human-IFN-β in FAT-7 bearing or non-tumor bearing immunocompetent rats did not result in acute organ toxicity or death.

Results: VSV-r-IFN-β replicated predominantly in tumors and a dose dependent anti-VSV antibody response was observed. Intratumoral or intravenous administration of VSV-IFN-β resulted in growth delay and improved survival compared with controls.

Conclusion: The above data confirm safety and feasibility of VSV-IFN-β administration in immunocompetent animals and support its clinical evaluation in advanced human head and neck cancer.

Keywords: biodistribution; preclinical studies; squamous cell carcinoma; syngeneic models; vesicular stomatitis virus.

FIGURE 1. Effects of VSV-r-IFN-β on FAT-7 cells in vitro

A, B. Dose Response. Cells were infected with VSV-r-IFN-β at different MOI’s and viable cell number (percent of control, A) and percent cytotoxicity (B) were determined. C. Time dependent cytotoxicity. Cells were infected with VSV-r-IFN-β and % cytotoxicity was determined at 24 (light grey bars), 48 (dark grey bars) and 72 hrs (black bars); * p= 0.14 (MOI=1 vs. control, 24 hours); ** p= 0.0008 (MOI=5 vs. control, 24 hours). D. Effects of VSV-r-IFN-β on cytotoxicity, during normoxia and hypoxia, at 48 hrs. * p= 0.09 (MOI=1 hypoxia vs. normoxia). Experiments in A-D were performed in triplicate, and repeated at least twice. E. In vitro viral replication. VSV-r-IFN-β titers were determined at 24 and 48 hours after infection by plaque forming assay (duplicate experiments). F. Western blot analysis of VSV-r-IFN-β expression in the FAT-7 conditioned medium (24 hours) after viral transduction at MOI=5 and 10.

FIGURE 2. Effects of VSV-m-IFN-β on SCC VII cells in vitro

A, B. Dose Response. Cells were infected with VSV-m-IFN-β at different MOI’s and viable cell number (percent of control, A) and percent cytotoxicity (B) were determined. C. Time dependent cytotoxicity. Cells were infected with VSV-m-IFN-β and % cytotoxicity was determined at 24 (light grey bars), 48 (dark grey bars) and 72 hrs (black bars). *p= 0.05 (MOI=1 vs. control, 24 hours); ** p= 0.0003 (MOI=5 vs. control, 24 hours). D. Effects of VSV-m-IFN-β on cytotoxicity, during normoxia and hypoxia, at 48 hrs. * p= 0.29. Experiments in A-D were performed in triplicate, and repeated at least twice. E. In vitro viral replication. VSV-m-IFN-β titers were determined at 24 and 48 hours after infection by plaque forming assay (duplicate experiments). F. Western blot analysis of VSV-m-IFN-β expression in the SCC VII conditioned medium (24 hours) after viral transduction at MOI=5 and 10.

FIGURE 3. In vitro cytotoxicity and viral replication of human or rat VSV-IFN-β on FAT-7 cells

FAT-7 cells were exposed to VSV-h-IFN-β or VSV-r-IFN-β at different MOIs and viability and cytotoxicity at 48 hours were assessed. A. Effects of VSV-h-IFN-β (solid lane) or VSV-r-IFN-β (broken lane) on viable cell number (% of control). B. Effects of VSV-h-IFN-β (black bars) or VSV-r-IFN-β (grey bars) on FAT-7 cytotoxicity at 48 hours. Experiments were performed in triplicate, and repeated at least twice C. In vitro viral replication of VSV-r-IFN-β (grey bars) and VSV-h-IFN-β (black bars) in FAT-7 Cells. Viral titers were determined by plaque forming assay at 48 hrs and at MOI-5 or 10 (duplicate experiments).

FIGURE 4. Tissue biodistribution and anti-VSV antibody production after intratumoral administration of VSV-r-IFN-β in immunocompetent rats

Fisher 344 rats bearing FAT-7 tumors in the neck area (as in materials and methods) were given one intratumoral injection of VSV-r-IFN-β (1×10¹⁰TCID₅₀ per rat), and tumors and tissues were harvested at day 2 (n=8) and 28/29 (n=6) after treatment. A. Recovery of VSV-r-IFN-β from tumor, brain and spleen tissues harvested from rats at day 2. Viral titers are displayed as TCID₅₀ per gram of tissue; arrow represents the assay’s limit of detection –LOD- (1.92 × 10² TCID₅₀/g tissue). B. Analysis of tissue biodistribution (assessed by quantitative RT-PCR of VSV-N RNA) after treatment with VSV-r-IFN-β. Total RNA was isolated from day 2 and 28/29 rats’ tumors and tissues. Data are displayed as copy number VSV-N RNA per μg or RNA. Tumor *: day 28/29 tumor sample. Dark line arrow=LOD, limit of detection; dotted line arrow=LOQ, limit of quantification). C. Anti-VSV antibody production. Anti-VSV ELISA was performed on day 28/29 serum samples.

FIGURE 5. Tissue biodistribution and anti-VSV antibody production after intratumoral administration of VSV-h-IFN-β in immunocompetent rats

Fisher 344 rats bearing FAT-7 tumors in the neck area (as in materials and methods) were given one intratumoral injection of VSV-h-IFN-β (1×10⁹TCID₅₀ per rat), and tumors tissues were harvested at day 2 (n=6) and day 28/29 (for antibody detection; n=6). A. Recovery of VSV-h-IFN-β from tumor, brain and spleen tissues harvested from rats at day 2. Viral titers are displayed as TCID₅₀ per gram of tissue; limit of detection –LOD- (arrow) = 1.92 × 10² TCID₅₀/g tissue. B. Analysis of tissue biodistribution (assessed by quantitative RT-PCR of VSV-N RNA) after treatment with VSV-h-IFN-β. Total RNA was isolated from day 2 rats’ tumors and tissues. Data are displayed as copy number VSV-N RNA per 0.2 μg or RNA. Dark line arrow=LOD, limit of detection; dotted line arrow=LOQ, limit of quantification). C. Anti-VSV antibody production. Anti-VSV ELISA was performed on day 28/29 serum samples.

FIGURE 6. In vivo effects of VSV-r-IFN-β or VSV-h-IFN-β on tumor growth and survival in immunocompetent rats bearing FAT-7 tumors

A. Average tumor volume (mm³) was determined in Fischer-344 (total n=40; 10 per group). Rats treated with escalating doses (5×10⁷; 5×10⁸ and 5×10⁹ PFUs of VSV-r-IFN-β or PBS. Treatment was initiated when tumors reached approximately 5 mm in diameter. *p<0.0001 (5×10⁸ vs. control). B. Effects of escalating doses of VSV-r-IFN-β on survival (measured by time to sacrifice, Kaplan-Meier Graph). **p=0.0008, 5×10⁸ vs. control. In separate experiments, tumor bearing rats (n=40) were treated with either intratumoral (1 vs. 2 treatments, n=10 per group) or intravenous (one treatment, n=10 per group) VSV-r-IFN-β, or PBS. Treatment was initiated when tumors reached approximately 5 mm in diameter. D. Average tumor volume (mm³). Tumor progression was significantly delayed in the treated groups, compared to controls (***p< 0.0001) B. Effects of virus treatment on survival (time to sacrifice; Kaplan-Meier Graph). **** p= 0.0003 (IT-1 vs. ctrl); p= 0.012 (IT-2 vs. ctrl); p=0.0084 (IV vs. ctrl).

FIGURE 7. In vivo effects of VSV-h-IFN-β in syngeneic rat squamous cell carcinoma

A. Average tumor volume (mm³) determined in Fischer-344 Rats (total n=30, 10 per group) treated with either intratumoral or intravenous VSV-h-IFN-β (5×10⁸ PFUs, for one dose) or PBS. Treatment was initiated when tumors reached approximately 5 mm in diameter. *p=0.0002 VSV-IT vs. ctrl; p=0.0004, VSV-IV vs. ctrl. B. Effects of intratumoral or intravenous VSV-h-IFN-β (5×10⁸ PFUs, for one dose) on survival (time to sacrifice). ** p<0.0001, IT/IV vs. control.