Toward high affinity VEGFR2 binding helix bundles

Objectives The purpose of this study is to use ribosome display to select high affinity three-helical bundle peptides that target vascular endothelial growth factor receptor type2 (VEGFR2). VEGFR2, an important component of tumor angiogenesis, is overexpressed in certain tumor blood vessels. VEGFR2 has been targeted for imaging using VEGF121 which is approximately 30kDa in size. We hypothesize that a smaller particle such as a 6kD sized VEGFR2 binding helical bundle will improve molecular imaging of VEGFR2 expression.

Methods The z-domain of protein A is used as the protein scaffold for the potential small protein tracer. Thirteen positions on the first two helices were mutated following the pioneering work by Nygren, Nord and company on Affibody. A 750bp double stranded DNA library encoding the Z-domain and C-terminal protein spacer were constructed for rabbit reticulocyte transcription/translation system (Promega). Maximal diversity of the library is 1.2x10¹¹. VEGFR2/FC (R&D systems) on streptavidin coated M280 beads (Dynal) is used for selection.

Results A small sampling of the assembled library sequences demonstrates randomization at the desired sites and approximately 50% of the sequences yield full length peptides. There is population enrichment for binding VEGFR2/FC up to the third round of selection. The RT-PCR product from the third round is cloned into expression vector and fused with maltose binding protein (MBP). Nine out of 75 clones isolated express mutant z-domain-MBP fusion. One of the clones has a Kd of 2x10^-7.

Conclusions A mutant of the z-domain of protein A with a weak affinity for VEGFR2/Fc fusion protein was found after three rounds of ribosome display. Affinity maturation is underway.

Research Support SNM Mallinckrodt Seed Grant 2007