Expression of anti-VEGF antibody together with anti-EGFR or anti-FAP enhances tumor regression as a result of vaccinia virotherapy

摘要

The tumor microenvironment plays an important role in tumor growth and progression. Here we demonstrate that vaccinia virus-mediated, constitutively expressed intratumoral antibodies against vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and fibroblast activation protein (FAP) significantly improved tumor regression and oncolytic virotherapy through suppression of angiogenesis, cell proliferation, and stromagenesis in virus-colonized tumors. In contrast to the tumor growth inhibition by the three tumor growth-inhibiting antibodies individually, when two of the three antibodies were expressed simultaneously by single vaccinia virus strains tumor regression was further enhanced. These findings strongly indicate that interference with the two tumor growth-stimulating mechanisms did in fact result in enhanced therapeutic efficacy in tumor xenograft models and may lead to an effective therapy in patients with cancer. prs.rt("abs_end"); Introduction In spite of great advances in anticancer treatments over the past 30 years, cancer remains the leading cause of death around the world. Overlooking the important role of tumor microenvironment (TME) in cancer growth and metastasis may be one of the reasons. 1 , 2 Angiogenesis and hyper-proliferation of cells in the stroma of tumors not only support the growth of cancer but also contribute to its development, for example, metastasis. Therefore, combining therapies that target cancer cells and the TME should result in greater therapeutic benefits than most of the current therapies that target cancer cells only. Factors within the TME such as vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and fibroblast activation protein (FAP) play crucial roles in cancer initiation and development. 3 , 4 and 5 The high-level expression of VEGF or EGFR correlates with poor prognosis in patients with breast, colon, lung, head and neck, and other cancers. 6 , 7 The anti-VEGF monoclonal antibody (mAb), bevacizumab (Avastin), was approved by the US Food and Drug Administration (FDA) in 2004 for the treatment of metastatic colon cancer and subsequently other metastatic cancers. In the same year, anti-EGFR mAb, cetuximab (Erbitux), was also approved by the FDA for the treatment of metastatic colon cancer. However, the clinical efficacy of Avastin and Erbitux has been somewhat limited 8 , 9 and 10 possibly due to poor tumor penetration and rapid clearance of the mAbs from the circulation, requiring the administration of high doses at frequent intervals and extensive durations, also making the therapies extremely costly. 11 Improvements in the pharmacodynamic properties of current mAb therapeutics and identification of additional functionalities targeting the TME could be greatly beneficial. For example, G6-31 is an improved anti-VEGF antibody derived from a phage display library with better binding affinity and enhanced therapeutic efficacy in animal models than Avastin. 12 To improve tumor penetration, a single-domain antibody of 15 kDa against EGFR from a llama has been recently developed (termed anti-EGFRVHH). 13 , 14 This llama nanobody was nonimmunogenic in mice and was proven to block binding of EGF to EGFR, thereby inhibiting EGFR signaling and showing the specific tumor targeting. 15 , 16 Anti-EGFRVHH is used for molecular imaging and therapeutic applications. 14 , 17 , 18 and 19 FAP (also known as seprase), a highly conserved protein, is richly expressed particularly in the stroma of aggressive cancers. 20 , 21 and 22 The high-level expression of FAP is correlated with cancer progression. 23 , 24 and 25 To date, no specific molecular inhibitor of FAP has been developed. 4 , 26 M036, a species-cross-reactive FAP-specific single-chain antibody (scAb), was isolated by sequential phage display and was shown to bind FAP on stromal cells of different human carcinomas and the murine host stroma in human tumor xenografts. 27 The therapeutic potential of M036 has not yet been evaluated. Combining the TME-targeted antiangiogenic and antiproliferative activities of these antibodies with a potent anticancer therapeutic in a form that could be simultaneously and efficiently administered was the goal of this research. The replication-competent oncolytic vaccinia virus (VACV) GLV-1h68 locates, replicates, and lyses tumor cells in human xenograft nude mouse models after administration of a single dose. 28 GLV-1h68 is currently in phase 1/2 clinical trials for the treatment of solid tumors. Additionally, recombinant VACVs can be genetically modified to express functional transgenes, including scAbs. We showed previously that VACVs expressing