Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy

Abstract

Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS.

Keywords: angiogenesis; antibody production; cancer; canine cancer cell lines; canine cancer therapy; canine soft tissue sarcoma (CSTS); oncolytic virus.

Figure 1

VEGF expression in MTH52c, ZMTH3, CT1258 or STSA-1 canine cancer cells under cell culture conditions. Each value represents the mean (n = 3) +/− standard deviations (SD).

Figure 2

Relative survival of MTH52c, ZMTH3, CT1258 or STSA-1 canine cancer cells after LIVP 6.1.1 or GLV-5b451 infection at an MOI of 0.1. Viable cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Mean values (n = 3) and standard deviations are shown as percentages of respective controls. The data represent two independent experiments.

Figure 3

Replication capacity of the vaccinia virus strains LIVP 6.1.1 (A) and GLV-5b451 (B) in different canine cancer cell lines. For the viral replication assay, MTH52c, ZMTH3, CT1258 or STSA-1 cells grown in 24-well plates were infected with either LIVP 6.1.1 or GLV-5b451 at an MOI of 0.1. Cells and supernatants were collected for the determination of virus titers at various time points. Viral titers were determined as pfu per ml in triplicates by standard plaque assay in CV-1 cell monolayers. Averages plus standard deviation are plotted. The data represent three independent experiments.

Figure 4

Expression of GLAF-2 protein in different canine cancer cells: (A) MTH52c; (B) ZMTH3; (C) CT1258 and (D) STSA-1. Western blot analysis of GLV-5b451-infected (MOI of 1.0; lines marked by *) or uninfected canine cancer cells. Protein fractions from cell lysates were isolated at 1, 24, 48, 72 and 96 h post virus infection and separated by SDS-PAGE. Western blot analysis was performed as described in material and methods. The position of GLAF-2 protein is marked by black arrow. M: PageRuler Prestained Protein Ladder # 26616 (Thermo Scientific, Bonn, Germany). The positions of the 35 and 25 kDa proteins are marked by $ symbol.

Figure 5

Interactions of purified GLAF-2 antibodies with canine, murine, and human VEGFs. Affinity and cross reactivity of GLAF-2 was demonstrated by ELISA. Equal concentrations of canine, murine, or human VEGF (100 ng/well) were coated on ELISA plates. Seven two-fold dilutions of purified GLAF-2 proteins ranging from 2000 ng/mL to 31.3 ng/mL were incubated with canine, murine and human VEGFs. PBS was used as negative control. For further ELISA experimental conditions see material and methods. ODs obtained for various concentrations of GLAF-2 against canine, murine and human VEGF were plotted. ELISA was repeated in three independent experiments. Each value represents the mean (n = 3) +/− standard deviations (SD).

Figure 6

Effects on tumor growth (A) and body weights (B) of virus- and mock-treated STSA-1 xenografted mice. (A) Groups of STSA-1-tumor-bearing nude mice (n = 7) were either treated with a single dose of 1 × 10⁷ pfu GLV-5b451 or LIVP 6.1.1 or with PBS (mock control). Statistical analysis was performed with a paired Student’s t-test (* p < 0.05); (B) Relative mean weight changes of STSA-1 cell xenografted mice after virus or PBS treatment. The data are presented as mean values +/− SD.

Figure 7

Presence of the scAb GLAF-2 (A) and analysis of vascular density in tumor tissues of virus- or PBS-injected STSA-1 xenograft mice at 17 dpvi (B–D). (A) Western blot analysis of GLV-5b451-infected STSA-1 tumors at 17 dpvi (lines 2–4). Line 1: Lysate of a LIVP 6.1.1-infected STSA-1 tumor (negative control). Each sample represents an equivalent of 1.5 mg tumor mass; (B,C) Visualization and analysis of vascular density using CD31 immunohistochemistry in LIVP 6.1.1, GLV-5b451 or PBS-treated tumors: (B) The vascular density was measured in CD31-labeled tumor cross-sections (n = 3 mice per group, 24 images per virus-injected mouse or 12 images per control PBS mouse) and presented as mean values +/− SD. (*** p < 0.001; ** p < 0.01;* p < 0.05 Student’s t-test); (C) Fluorescence intensity of the CD31 signal of blood vessels. The fluorescence intensity of the CD31-labelling represents the average brightness of all vessel-related pixels and was determined as described in materials and methods. Shown are the mean values +/− standard deviations. Statistical analysis was performed with a two-tailed unpaired Student’s t-test (*** p < 0.001; ** p < 0.01; * p < 0.05); (D) Representative tumor sections labeled with anti-CD31 antibody (red) and anti-vaccinia virus (VV) antibody (green). Scale bars: 150 mm.