Identification of a high affinity TAG-72 binding peptide by phage display selection

Purpose: Phage display was used to select novel peptides that specifically bind the TAG-72 antigen and with properties suitable for imaging TAG-72 positive cancers. Results: After three rounds of selection against TAG-72 and using two different elution conditions including a long elution, the consensus sequences FRE RCD KHP QKC TKF L and DPR HCQ KRV LPC PA W L were expressed on phages G3-15 and T3-15 respectively. ELISA, fluorescence-activated cell sorting analysis and fluorescence microscopy provided evidence that both phages specifically bound TAG-72 in vitro. Both peptides are stable in 37°C serum. By a cell binding competition assay, the IC₅₀ for T3-15 was measured as 0.29 nM and therefore 36-fold higher affinity than G3-15 at 10.32 nM. The biodistribution in mice carrying LS-174T tumors in one thigh were similar for both ^99mTc-peptides at 30 min, but at 90 min the ^99mTc-T3-15 peptide accumulated almost three times higher in the tumor. The SPE CT/CT images were consistent with the biodistribution results. Procedures: The f88-4/Cys6 phage library and two different elution conditions were used to identify two new higher affinity binding peptides for the TAG-72 antigen. One, was a single brief elution with pH 2.2 glycine buffer and the second began with the glycine elution but was followed with a longer elution with Tris buffered saline (TBS) at pH 7.4. The phages that bound TAG-72 were evaluated by fluorescence-activated cell sorting analysis using TAG-72 positive LS-174T cells and confirmed by immunofluorescence imaging. The consensus peptides displayed on the selected phages were synthesized and conjugated with NHS-MAG3 for radiolabeling with ^99mTc. The IC₅₀ for TAG-72 binding was evaluated by cell binding competition in vitro while binding affinity was evaluated in vivo by necropsy and SPE CT/CT imaging in a tumor mouse model. Conclusion: We have identified a peptide with a sub nanomolar inhibition constant for the TAG-72 antigen that may have applications in cancer imaging.