Identification of a high affinity TAG-72 binding peptide by phage display selection
Department of Radiology; Department of Medicine, Divison of Hematology/Oncology
Amino Acid Sequence; Animals; Antigens, Neoplasm; Cell Line, Tumor; Colonic Neoplasms; Glycoproteins; HT29 Cells; Humans; Male; Mice; Mice, Nude; Molecular Sequence Data; Peptide Library; Peptides; Protein Binding; Technetium; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Xenograft Model Antitumor Assays
Cancer Biology | Neoplasms | Oncology | Radiology | Therapeutics
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 FRERCDKHPQKCTKFL and DPRHCQKRVLPCPAWL 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 37oC 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 SPECT/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 SPECT/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 application in cancer imaging.
phage display, TAG-72 antigen, peptide, colon cancer tumor cell LS-174T
DOI of Published Version
Cancer Biol Ther. 2011 Jan 1;11(1):22-31. DOI: 10.4161/cbt.11.1.13797. Link to article on publisher's website
Cancer biology and therapy
Xiao N, Cheng D, Wang Y, Chen L, Liu X, Dou S, Liu G, Liang M, Hnatowich DJ, Rusckowski M. (2011). Identification of a high affinity TAG-72 binding peptide by phage display selection. Radiology Publications and Presentations. https://doi.org/10.4161/cbt.11.1.13797. Retrieved from https://escholarship.umassmed.edu/radiology_pubs/49