Many therapeutics and diagnostics are being developed that employ molecular targeting strategies to bind upregulated receptors and specifically localize the desired molecules into diseased tissue. While targeted ligands often provide dramatic improvements over their untargeted analogues, target receptors are also present in healthy tissues thus limiting the systemic specificity of the targeting strategy. In an effort to improve targeting specificity, we have developed a unique method for engineering protease-activated binding ligands, or proligands. First, we engineered a tumor-targeting peptide that selectively binds vascular endothelial growth factor (VEGF) only after exposure to tumor associated proteases, matrix metalloproteases (MMP), that are present in cancerous tissue. Bacterial peptide display libraries were constructed and sorted using flow cytometry to select an inhibitor to a VEGF binding peptide. This inhibitor is fused to the VEGF binding peptide via an MMP substrate, and the resulting propeptide construct exhibits a 5-fold improvement in binding affinity after exposure to protease. We have extended this approach to create pro-antibodies, wherein a peptide epitope masking the antigen binding site is recombinantly tethered to single chain antibodies (ScFv) until cleavage of a protease susceptible linker. With the goal of more specifically targeting inflammatory processes in atherosclerosis and cancer, MMP activated scFvs were constructed that bind vascular cell adhesion molecule-1 (VCAM-1). The binding properties of these proantibodies are currently being evaluated. This method may prove useful for improving systemic targeting specificity of a variety of protein therapeutic and diagnostic agents.

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