r84, a Novel Therapeutic Antibody against Mouse and Human VEGF with Potent Anti-Tumor Activity and Limited Toxicity Induction Laura A. Sullivan1, Juliet G. Carbon 1 , Christina L. Roland1, Jason E. Toombs1, Mari Nyquist-Andersen 2 , Anita Kavlie2, Kyle Schlunegger3, James A. Richardson 4 , Rolf A. Brekken1,5* 1 Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 2 Affitech AS, Oslo Research Park, Oslo, Norway, 3 Peregrine Pharmaceuticals, Inc., Tustin, California, United States of America, 4 Departments of Pathology and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 5 Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America Abstract Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF:VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF:VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors. Citation: Sullivan LA, Carbon JG, Roland CL, Toombs JE, Nyquist-Andersen M, et al. (2010) r84, a Novel Therapeutic Antibody against Mouse and Human VEGF with Potent Anti-Tumor Activity and Limited Toxicity Induction. PLoS ONE 5(8): e12031. doi:10.1371/journal.pone.0012031 Editor: Andrei L. Gartel, University of Illinois at Chicago, United States of America Received June 1, 2010; Accepted July 13, 2010; Published August 6, 2010 Copyright: 2010 Sullivan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by a sponsored research agreement from Peregrine Pharmaceuticals (to R. A. Brekken), institutional funding from the Simmons Comprehensive Cancer Center, the Randall Bridwell Lung Cancer Research Grant from Uniting Against Lung Cancer (to R. A. Brekken, www. UnitingAgainstLungCancer.org), a development award from the UT SPORE in lung cancer (P50 CA070907) (to R. A. Brekken), the Effie Marie Cain Scholarship in Angiogenesis Research (to R. A. Brekken) and Affitech AS (M. Nyquist-Andersen, A. Kavlie). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: R. A. Brekken is a consultant for, has equity interest in, and is a recipient of a sponsored research grant from Peregrine Pharmaceuticals and Affitech AS. R. A. Brekken is also an author of a patent on technology that was used to develop the antibody r84 by Peregrine Pharmaceuticals and Affitech AS. M. Nyquist-Andersen and A. Kavlie are employees of Affitech AS, and K. Schlunegger is an employee of Peregrine Pharmaceuticals, which supported these studies by providing r84, mcr84, and funding. Peregrine and Affitech did not participate in the planning, execution, or interpretation of the experiments. The authors affirm that they will adhere to the PLoS data sharing statement. * E-mail: rolf.brekken@utsouthwestern.edu Introduction Angiogenesis is a tightly regulated process that is essential during growth, wound healing and development, as well as cancer growth, progression and metastasis [1–2]. A key stimulant of angiogenesis is vascular endothelial growth factor-A (VEGF). VEGF induces endothelial cell survival, proliferation, and migration its predomi- nant signaling receptor, VEGF receptor 2 (VEGFR2). Tumor associated macrophages also express VEGFR2 and selective blockade of VEGFR2 is able to decrease macrophage infiltration into tumors [3]. VEGF signaling through VEGF receptor 1 (VEGFR1) remains unclear, although it is thought to have effects on hematopoiesis, vascular permeability, and monocyte migration. Importantly, there is elevated expression of VEGF, VEGFR1, and VEGFR2 within tumors, providing a therapeutic target. In fact targeting VEGF has lead to the development of anti-angiogenic therapies such as sunitinib malate (SutentH, SU11248, Pfizer, Inc.), sorafenib (NexavarH, BAY 43-9006, Bayer Pharmaceuticals Corp.), bevacizumab (AvastinH, Genentech), IMC-1121b (ramucirumab, ImClone), VEGF-Trap (aflibercept, Regeneron) and 2C3 [2,4–6]. Sunitinib and sorafenib are small molecule inhibitors of multiple receptor tyrosine kinases (RTKs) including the VEGF receptors. These drugs have been FDA-approved for the treatment of renal cell carcinoma, gastrointestinal stromal tumors (GIST) (sunitinib), and unresectable hepatocellular carcinoma (sorafenib) [2,7–8]. Bevacizumab is a humanized monoclonal anti-VEGF antibody that inhibits VEGF from binding to and signaling through VEGFR1 and VEGFR2. Bevacizumab is approved in combina- tion with cytotoxic chemotherapy for the treatment of colorectal cancer, non-small cell lung cancer (NSCLC), and breast cancer, as PLoS ONE | www.plosone.org 1 August 2010 | Volume 5 | Issue 8 | e12031