Preclinical development of a potent VEGFR-2 antagonist based on a novel protein scaffold (AdNectin)
Authors: Roni Mamluk, Irvith M. Carvajal, Janna M. Bates, Dimitry M. Kamen, Brent A. Morse, Steve G. Kovats, Ai-Ching Lim, Jochem Gokemeijer, Michael J. Storek, Joonsoo Lee, Jack Wang, Alan S. Harris, Gordon Wong
Journal: Cancer Research (2005)

3037   Shortcomings of traditional antibody technology have prompted the development of antagonists using addressable fibronectin proteins (AdNectins). These novel antagonists are based on the 10th Type III domain of human fibronectin (∼10 kDa). 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. Activation of VEGFR-2 stimulates multiple endothelial cell functions (e.g., migration, proliferation, and survival) and induces angiogenesis and blood vessel permeability. The anti-VEGFR-2 AdNectin, CT322, is a 10 kDa monomeric protein that is highly stable at 37°C. CT322 binds human and mouse VEGFR-2-Fc with affinities of 0.2 nM and 4 nM, respectively. Low levels of CT322 (130 pM) were sufficient to completely block VEGF-A induced VEGFR-2 phosphorylation and ERK1/2 activation in human primary endothelial cells. CT322 efficiently blocked VEGF-induced human primary endothelial cell proliferation, migration and tube formation. Moreover, CT322 blocked both VEGF-C and VEGF-D induced activation of VEGFR-2. Together these data indicate that CT322 is a potent antagonist of VEGFR-2 in endothelial cells. The pharmacokinetic profile for CT322 was determined in rats using  125 I-labelled CT322. CT322 was rapidly cleared (70 ml/hr/kg) via the kidneys. Addition of a single, branched 40 kDa polyethylene glycol (PEG40) reduced the clearance rate to 5.2 ml/hr/kg leading to 10 fold increase in bioavailability as calculated by AUC of 14.6 and 193 hr*μg/ml for CT322 and CT322-PEG40, respectively. No accumulation of CT322-PEG40 in normal tissues was observed. A classical Miles assay for microvessel permeability was used to determine whether CT322 can also block VEGFR-2  in vivo . Mice were pre-treated by a single i.p. injection of CT322-PEG40 4 hours prior to the experiment. CT322-PEG40 inhibited VEGF-induced vessel permeability at 5 mg/kg dose indicating an effective blockage of VEGFR-2 activity  in vivo . The effect of CT322-PEG40 in various mouse and human xenografts tumor models was subsequently evaluated. CT322-PEG40 significantly inhibited tumor growth in the various models at dose range of 10 - 40 mg/kg suggesting a potential therapeutic use. In summary: We have developed a receptor antagonist based on a novel alternative protein scaffold that can be produced in  E. coli  and has high target specificity and affinity as well as favorable biophysical properties. Although CT322 is intended to block human VEGFR-2 (KDR), it was designed to also be effective in blocking murine VEGFR-2 (Flk1). Consequently, CT322 has been shown to be very effective in inhibiting tumor growth in multiple pre-clinical models.