Abstract
Epithelial ovarian cancer is the deadliest gynecological malignancy. The lack of effective treatments highlights the need for novel therapeutic interventions. The aim of this study was to investigate whether sustained adeno-associated virus (AAV) vector-mediated expression of vascular normalizing agents 3TSR and Fc3TSR and the antiangiogenic monoclonal antibody, Bevacizumab, with or without oncolytic virus treatment would improve survival in an orthotopic syngeneic mouse model of epithelial ovarian carcinoma. AAV vectors were administered 40 days post-tumor implantation and combined with oncolytic avian orthoavulavirus-1 (AOaV-1) 20 days later, at the peak of AAV-transgene expression, to ascertain whether survival could be extended. Flow cytometry conducted on blood samples, taken at an acute time point post-AOaV-1 administration (36 h), revealed a significant increase in activated NK cells in the blood of all mice that received AOaV-1. T cell analysis revealed a significant increase in CD8+ tumor specific T cells in the blood of AAV-Bevacizumab+AOaV-1 treated mice compared to control mice 10 days post AOaV-1 administration. Immunohistochemical staining of primary tumors harvested from a subset of mice euthanized 90 days post tumor implantation, when mice typically have large primary tumors, secondary peritoneal lesions, and extensive ascites fluid production, revealed that AAV-3TSR, AAV-Fc3TSR+AOaV-1, or AAV-Bevacizumab+AOaV-1 treated mice had significantly more tumor-infiltrating CD8+ T cells than PBS controls. Despite AAV-mediated transgene expression waning faster in tumor-bearing mice than in non-tumor bearing mice, all three of the AAV therapies significantly extended survival compared to control mice; with AAV-Bevacizumab performing the best in this model. However, combining AAV therapies with a single dose of AOaV-1 did not lead to significant extensions in survival compared to AAV therapies on their own, suggesting that additional doses of AOaV-1 may be required to improve efficacy in this model. These results suggest that vectorizing anti-angiogenic and vascular normalizing agents is a viable therapeutic option that warrants further investigation, including optimizing combination therapies.
Highlights
Ovarian cancer is the seventh most commonly diagnosed cancer among women in the world [1] and accounts for more deaths than any other cancer of the female reproductive system [2]
The heavy (KX119517.1) and light chains of Bevacizumab (KX119516.1), separated by a self-cleaving F2A peptide, were synthesized as a full length human IgG antibody and cloned into the KpnI-XbaI site of pACASI-MCS-WPRE which contains a strong constitutive CASI promoter [57], a WPRE element [58] and an SV40 polyA signal surrounded by AAV2 inverted terminal repeats. 3TSR from human TSP-1 (NM_003246.4) synthesized to contain a 6xHis tag at the C-terminus was cloned into the
The concentration of 3TSR in the serum and the peritoneal cavity was markedly lower than Fc3TSR or Bevacizumab (Figure 1B,E) and was only detectable in the serum of mice that received associated virus (AAV)-3TSR i.m., where it peaked on day 56 at 1.4 ± 1.5 μg/mL and was below the level of detection by day 142 (Figure 1B)
Summary
Ovarian cancer is the seventh most commonly diagnosed cancer among women in the world [1] and accounts for more deaths than any other cancer of the female reproductive system [2]. Treatment modalities that block VEGF and its pro-angiogenic functions have been validated in various types of human cancers and neoplastic tissues [7]. The an anti-VEGF monoclonal antibody (mAb), Avastin® (bevacizumab), is an approved treatment for multiple types of cancers including lung, colon, glioblastoma, and renal-cell carcinoma [8]. When VEGF-A cannot bind to its receptors, tumor blood supply and interstitial pressure are reduced, while chemotherapy delivery and vascular permeability are increased [10]. Bevacizumab treatment increases progression-free survival in women with ovarian cancer [11,12,13], transient and low peritoneal drug levels can influence treatment efficacy [14]. Since tumor cells constantly express VEGF [15], a persistent delivery of bevacizumab may be needed to prevent revascularization associated with anti-VEGF therapy withdrawal. Using a gene therapy vector to constitutively express Bevacizumab represents one way to mediate sustained expression and reduce the peaks and troughs associated with systemic delivery of recombinant mAbs [18,19]
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