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

UMMS Affiliation

Department of Radiology; Department of Medicine, Divison of Hematology/Oncology

Publication Date


Document Type



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

Journal/Book/Conference Title

Cancer biology and therapy

Related Resources

Link to Article in PubMed

PubMed ID