Rapid identification and development of αvβ6 -targeting peptides as PET imaging agents using fluorescent cell-based on-bead screening

Objectives Combinatorial libraries such as one-bead-one-compound (OBOC) peptide libraries have facilitated the rapid identification of receptor targeting ligands.1 We and others are now using OBOC libraries to develop PET imaging agents, including the development of [18F]fluorobenzoyl ([18F]FB) peptides to target the αvβ6 -integrin.2 This cell surface receptor is overexpressed in numerous aggressive cancers.3 Using an on-bead cell-screening assay, which employs subsequent rounds of screening with αvβ6 -positive and αvβ6 -negative cells, we identified αvβ6 -binding peptides. Unfortunately, a major drawback of this approach is the lengthy screening process. In this work, we present an expedited screening process employing fluorescent cells. This one-step fluorescent screening assay was evaluated on a XXDLXXLXK(FB) peptide library, which features the known αvβ6 -binding motif DLXXL (X=random L-amino acid) as well as the widely used fluorobenzoyl (FB) moiety for the subsequent development of 18F-peptides.4

Methods A 9-mer XXDLXXLXK(FB) peptide library was assembled on TentaGel resin (1g, ~3,000,000 beads) using standard Fmoc chemistry. Small aliquots (50mg, ~150,000 beads) were incubated with a mixture of red fluorescent DX3 ITGβ6 mCherry (αvβ6 -positive) and green fluorescent DX3 EmGFP (αvβ6 -negative) cells for 3 hours and evaluated under a fluorescent microscope. Beads that were covered only by red (i.e. αvβ6 -positive) cells were isolated; these beads were subsequently characterized by Edman sequencing. Following synthesis of the identified peptides, competitive binding ELISA was performed with purified integrin to evaluate binding specificities for the target αvβ6 -integrin as well as closely related αv-integrins.

Results To date, 185 αvβ6 -selective beads were isolated from the peptide library. As proof of concept, five beads were selected for sequencing and binding specificities toward αvβ6, αvβ3 and αvβ5 integrins were evaluated using ELISA. The peptides tested show strong binding to the αvβ6 -integrin (IC50 values ranging from 0.3 to 6.6 nM) with high selectivity ratios over αvβ5 and αvβ3 (1,000:1 and 10,000:1, respectively).

Conclusions Using fluorescent cells, we have streamlined the OBOC library on-bead screening approach. One-step screening for αvβ6 -targeting peptides and preliminary sequencing to date yielded four peptides with strong affinity and high selectivity for the αvβ6 -integrin. Further work is underway to assess the potential of these 18F-peptides in vitro in cell binding and serum stability studies. The lead peptide, based on affinity and stability, will be tested in vivo. We anticipate this screening approach will be readily applicable to other target proteins for the development of novel imaging agents. Research Support: This research was supported by NIH 1RC4EB01 2836-01 and the Office of Science, US Department of Energy DE-SC0002061