Large Molecule Therapeutics Novel Glycosylated VEGF Decoy Receptor Fusion Protein, VEGF-Grab, Efficiently Suppresses Tumor Angiogenesis and Progression Jung-Eun Lee 1,2 , Chan Kim 1,3 , Hannah Yang 1 , Intae Park 1 , Nuri Oh 1,2 , Serenus Hua 4 , Haneul Jeong 4 , Hyun Joo An 4 , Sun Chang Kim 2 , Gyun Min Lee 2 , Gou Young Koh 1,2 , and Ho Min Kim 1 Abstract Antiangiogenic therapies targeting VEGFA have been common- ly used in clinics to treat cancers over the past decade. However, their clinical efficacy has been limited, with drawbacks including acquisition of resistance and activation of compensatory path- ways resulting from elevated circulating VEGFB and placental growth factor (PlGF). To bypass these disadvantages, we devel- oped a novel glycosylated soluble decoy receptor fusion protein, VEGF-Grab, that can neutralize VEGFA, VEGFB, and PlGF. VEGF- Grab has the second and third immunoglobulin (Ig)-like domains of VEGF receptor 1 (VEGFR1) fused to IgG1 Fc, with three potential glycosylation sites introduced into the third Ig-like domain of VEGF-Grab by mutagenesis. Compared with VEGF- Trap, VEGF-Grab showed more potent decoy activity against VEGF and PlGF, mainly attributed to the VEGFR1 backbone. Most importantly, the negatively charged O-glycans attached to the third Ig-like domain of VEGFR1 counterbalanced the origi- nally positively charged VEGFR1 backbone, minimizing nonspe- cific binding of VEGF-Grab to the extracellular matrix, and result- ing in greatly improved pharmacokinetic profile. These advance- ments led to stronger and more durable antiangiogenic, antitu- mor, and antimetastatic efficacy in both implanted and spontaneous tumor models as compared with VEGF-Trap, while toxicity profiles were comparable with VEGF-Trap. Collectively, our results highlight VEGF-Grab as a promising therapeutic can- didate for further clinical drug development. Mol Cancer Ther; 14 (2); 1–10. Ó2014 AACR. Introduction VEGFA is a critical regulator of tumor angiogenesis, mainly through the activation of its primary receptor, VEGF receptor 2 (VEGFR2; refs. 1–3). VEGFA is expressed in most tumor cells and corresponding stromal cells throughout tumor progression, where- as VEGFR2 is highly expressed in growing tumor vessels, leading to the formation of structurally and functionally malformed tumor blood vessels (1, 4). VEGFA specifically binds to the second immunoglobulin (Ig) homology domain (D2) of the extracellular region of VEGFR2, resulting in activation of proangiogenic signal- ing (5). For the past decade, much effort has been devoted to targeting this VEGFA/VEGFR2 signaling pathway using monoclo- nal antibodies, soluble decoy receptor fusion proteins, and small- molecular inhibitors in patients with cancer (6–9). Although the current therapeutic blockade of VEGFA/VEGFR2 signaling pro- vides clinical benefits, the anticancer effect is transient, eventually giving rise to acquired resistance through the activation of alter- native proangiogenic pathways and further recruitment of proan- giogenic cells such as tumor-associated macrophages (TAM; refs. 10–12). These limitations highlight current unmet needs in antiangiogenic cancer treatment strategies, which must be addressed for successful therapy development. VEGFA also binds to VEGFR1 with higher affinity (<10–20 pmol/L) than VEGFR2 (<100–125 pmol/L; ref. 13). In addition, VEGFR1 is a receptor for other proangiogenic ligands, VEGFB and placental growth factor (PlGF), which have recently been highlighted as alternative targets for antiangiogenic therapy (14–17). Because of its ability to bind multiple proangiogenic ligands, VEGFR1 has been considered as a potential backbone for the development of a novel decoy receptor fusion protein for therapeutic purposes. However, the efficiency of a decoy receptor fusion protein, which consisted of the first three Ig domains of VEGFR1 fused with the Fc region of IgG1 (VEGFR1-Fc), proved unsatisfactory due to nonspecific binding to the extracellular matrix (ECM) attributed to the abundant positively charged residues in the third Ig domain (VEGFR1 D3) and its high isoelectric point (pI; ref. 7). Nonetheless, this finding inspired the invention of VEGF-Trap (Aflibercept, Regeneron), consisting of VEGFR1 D2 and VEGFR2 D3 fused to IgG1 Fc. By switching VEGFR1 D3 to VEGFR2 D3, net pI of VEGF-Trap was decreased, resulting in less ECM bindings and an improved pharmacokinetic (PK) profile compared with VEGFR1-Fc (7). However, because 1 Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea. 2 Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea. 3 Division of Medical Oncology, Department of Internal Medicine,Yonsei University College of Medicine, Seoul, Korea. 4 Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). J.-E. Lee and C. Kim contributed equally to this article. Corresponding Authors: Ho Min Kim, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea. Phone: 82- 42-350-4244; Fax: 82-42-350-4240; E-mail: hm_kim@kaist.ac.kr; and Gou Young Koh, E-mail: gykoh@kaist.ac.kr doi: 10.1158/1535-7163.MCT-14-0968-T Ó2014 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org OF1 Research. on January 30, 2015. © 2014 American Association for Cancer mct.aacrjournals.org Downloaded from Published OnlineFirst December 22, 2014; DOI: 10.1158/1535-7163.MCT-14-0968-T