Improving pharmacokinetics of an ErbB2-targeted peptide by phage display affinity maturation

Objectives The ErbB2 receptor is well known for its oncogenic properties in several cancers. Previously, a peptide was selected from a phage library to target ErbB2. The radiolabeled peptide bound to ErbB2-expressing human breast cancer xenografts; however, moderate tumor uptake and high non-target organ accumulation prevented clinical utility. In vitro and in vivo phage display selections using an ErbB2-targeting peptide microlibrary were performed in an attempt to improve tumor uptake and pharmacokinetics.

Methods A novel phage library was designed to display the 1st generation peptide flanked by random amino acids (nnnnnKCCYSLnnnn) for parallel selections against purified ErbB2 and ErbB2-expressing tumors. Phage were evaluated for the ability to bind human breast carcinoma cells and peptides from phage demonstrating breast cancer affinity greater than the parent phage were synthesized. Peptide specificity and affinity was analyzed by fluorescent microscopy, flow cytometry and ELISA. Second generation peptides with the highest ErbB2 specificity and affinity were synthesized as DOTA-conjugates, radiolabeled with 111-In, characterized in vitro and analyzed in breast tumor-bearing mice.

Results Peptides with affinities greater than that observed for the original peptide (KD~500 nM) were obtained. Tumor uptake, in general, was less in 2nd generation compared to 1st generation peptides. The leading 2nd generation peptide had similar tumor uptake (0.70%ID/g) with respect to the 1st generation peptide (0.78%ID/g); however kidney uptake (4.6%ID/g) was reduced in comparison to the parent peptide (5.8%ID/g). Non-tumor uptake in other organs was significantly reduced in comparison to the 1st generation peptide, as evidenced by increased tumor to muscle ratios (14:1 vs. 8.7:1) in 2nd and 1st generation peptides, respectively.

Conclusions Although affinity maturation did not improve tumor uptake, reduction in non-target organ uptake resulted in 2nd generation peptides with improved pharmacokinetics in comparison to the 1st generation peptide.

Research Support VA Merit to SLD