Development Of A Vegfr-2 Antagonist Based On A Novel Protein Scaffold (Adnectin)

3150 Background: Shortcomings of traditional antibody technology have prompted the development of antagonists using addressable fibronectin proteins (AdNectins). The benefits of AdNectins include the ability to rapidly identify high affinity binders with tailored binding kinetics, as well as the ability to produce these proteins by economic E. coli-based production Methods: Using this technology an AdNectin antagonist to VEGFR-2 was developed. VEGFR-2 was chosen as a target because of its well validated role in angiogenesis. Methods: The anti-VEGFR-2 AdNectin, CT322, was evaluated pre-clinically for its biophysical characteristics, ability to block VEGFR-2 activity on endothelial cells, pharmacokinetics, inhibition of VEGF activity in vivo, and effects on tumor progression in mouse models. Results: CT322, is a PEGylated 10 kDa monomeric protein that is highly stable at 37°C. CT322 binds human and mouse VEGFR-2 with affinities of 0.2 nM and 4 nM, respectively. CT322 efficiently blocked VEGF-induced endothelial cell proliferation, migration and tube formation. Pharmacokinetic in rats: UnPEGylated CT322 was rapidly cleared (70 ml/hr/kg) via the kidneys. Addition of a 40 kDa polyethylene glycol reduced the clearance rate to 5.2 ml/hr/kg leading to 10 fold increase in bioavailability. No accumulation of CT322 in normal tissues was observed. In vivo, CT322 inhibited VEGF-induced vascular permeability at 5 mg/kg dose indicating an effective blockage of VEGFR-2 activity in mice. CT322 significantly inhibited tumor growth in a range of mouse and human xenografts tumor models at dose range of 10 - 40 mg/kg (i.p. 3x week). Conclusions: CT322 is a VEGFR-2 antagonist based on a novel alternative protein scaffold and has high target specificity and affinity, as well as favorable biophysical properties. CT322 block human VEGFR-2 and murine VEGFR-2 (Flk1). Consequently, CT322 has been shown to be very effective in inhibiting tumor growth in multiple pre-clinical models. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Compound Theraeutics, Gokemeijer