Anti-VEGF single-chain antibody GLAF-1 encoded by oncolytic vaccinia virus significantly enhances antitumor therapy

Abstract

We previously reported that the replication-competent vaccinia virus (VACV) GLV-1h68 shows remarkable oncolytic activity and efficacy in different animal models as a single treatment modality and also in combination with chemotherapy [Yu YA, et al. (2009) Mol Cancer Ther 8:141-151]. Here, we report the construction of 3 VACV strains encoding GLAF-1, a previously undescribed engineered single-chain antibody (scAb). This unique scAb is transcribed from 3 vaccinia promoters (synthetic early, early/late, and late) and directed against both human and murine VEGFs. The expression of GLAF-1 was demonstrated in cell cultures. Also, the replication efficiency of all GLAF-1-expressing VACV strains in cell culture was similar to that of the parental GLV-1h68 virus. Successful tumor-specific delivery and continued production of functional scAb derived from individual VACV strains were obtained in tumor xenografts following a single intravenous injection of the virus. The VACV strains expressing the scAb exhibited significantly enhanced therapeutic efficacy in comparison to treatment of human tumor xenografts with the parental virus GLV-1h68. This enhanced efficacy was comparable to the concomitant treatment of tumors with a one-time i.v. injection of GLV-1h68 and multiple i.p. injections of Avastin. Taken together, the VACV-mediated delivery and production of immunotherapeutic anti-VEGF scAb in colonized tumors may open the way for a unique therapy concept: tumor-specific, locally amplified drug therapy in humans.

Fig. 1.

Combination therapy of s.c. PANC-1 tumors with GLV-1h68 virus and Avastin. Tumor-bearing mice (n = 8 per group) were treated with the virus alone (5 × 10⁶ pfu/mouse), with Avastin alone (5 mg/kg i.p. twice weekly for 5 weeks), with the virus initially (5 × 10⁶ pfu/mouse) followed by Avastin treatment (5 mg/kg twice weekly for 5 weeks) 13 days later, or with no treatment. The up-pointing arrow indicates the time of virus injection. The down-pointing arrows indicate the beginning and end of Avastin treatment. Statistical analysis was performed using one-way ANOVA (**P < 0.01, *P < 0.05). Stars indicate the comparison of the GLV-1h68 group with the Avastin + GLV-1h68 group.

Fig. 2.

Viral replication in A549 cells resulted in over-expression of the GLAF-1 scAb protein. (A) Schematic representation of the GLAF-1 scAb construct. (B) VACV constructs and marker genes. The LIVP WT virus strain was used for the generation of modified GLV-1h68 according to Zhang et al. (8). The GLV-1h68 virus was used for the construction of GLV-1h107, GLV-1h108, and GLV-1h109. p11, VACV p11 late promoter; pSEL, VACV SEL promoter; pSE, VACV SE promoter; pSL, VACV SL promoter; p7.5, VACV 7.5K early/late promoter. (C) Replication efficiency was determined after infection of CV-1 or A549 cells with VACV strains at an MOI of 0.01. Statistical analysis was performed using one-way ANOVA (**P < 0.01, *P < 0.05). Stars indicate comparison of the GLV-1h68 group with the groups of the unique viruses. (D) Expression of GLAF-1 in tumor cells in vitro. A549 cells were infected with VACV strains at an MOI of 1, and protein fractions from cell lysate and culture supernatant were isolated and separated by SDS/PAGE. Western blot analysis was performed using an anti-DDDDK antibody. The line shows the protein of the expected size of 30 kDa.

Fig. 3.

GLAF-1 expression in GLV-1h108– and GLV-1h109–colonized tumors leads to enhanced tumor therapy and inhibition of BVD in mouse xenografts. (A) Therapeutic effect of VACV strains delivering GLAF-1 on tumor growth in DU-145 tumor-bearing nude mice (n = 8). The up-pointing arrow indicates the time of virus injection. The down-pointing arrows indicate the beginning and end of Avastin treatment. Statistical analysis was performed using one-way ANOVA (** P< 0.01, *P < 0.05). Stars indicate comparison of the GLV-1h68 group with the Avastin + GLV-1h68 group (gray) and the GLV-1h108 group (black). (B) Therapeutic effect of VACV strains delivering GLAF-1 on tumor growth in A549 tumor-bearing nude mice (n = 7–8). Statistical analysis was performed using one-way ANOVA (**P < 0.01, *P < 0.05). Stars indicate comparison of the GLV-1h68 group with the GLV-1h108 group (gray) and the GLV-1h109 group (black). (C) Quantitative analysis of BVD in DU-145 tumors 21 days after VACV infection. Data are given as means of cell number per high-power field (×200 magnification, n = 3) (D) Expression of GLAF-1 in sera and correlation of tumor volume changes of VACV-infected A549 tumor-bearing mice. Tumor fluid and retro-orbital blood samples were taken at days 7, 21, and 37 after virus injection from the same mice. Tumor growth was monitored by caliper measurements. Protein amounts were quantitatively determined by ELISA with VEGF-precoated plates. Experimental groups contained 4 animals each.

Fig. 4.

Effect of virus treatment on tumor vasculature in DU-145 tumors. Tumors were excised at day 21 p.i., fixed, sectioned, and stained for CD31 to label endothelial cells (red). GFP expression indicates viral infection (green). All images are representative examples. (Scale bar: 100 μm.)