Pharmacokinetically Stabilized Cystine Knot Peptides That Bind Alpha-v-Beta-6 Integrin with Single-Digit Nanomolar Affinities for Detection of Pancreatic Cancer

Purpose: Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin alpha(v)beta(6), a cell surface receptor being evaluated as a novel clinical biomarker. Experimental Design: To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3-6 nmol/L) to integrin alpha(v)beta(6) The binders do not cross-react with related integrin alpha(v)beta(5), integrin alpha(5)beta(1), or tumor-angiogenesis-associated integrin, alpha(v)beta(3). Results: Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin alpha(v)beta(6). Clinically relevant tumor-to-muscle ratios of 7.7 +/- 2.4 to 11.3 +/- 3.0 were achieved within I hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting alpha(v)beta(6)-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3). Conclusions: We have engineered highly stable cystine knot peptides with potent and specific integrin a. alpha(v)beta(6)-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging. Clin Cancer Res; 18(3); 839-49. (C) 2011 AACR.