title: Identification of a high affinity TAG-72 binding peptide by phage display selection.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  We have identified a peptide with a sub nanomolar inhibition constant for the TAG-72 antigen that may have application in cancer imaging.
authors.affiliation: University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Xiao
authors.forename: Dengfeng
authors.initials: D
authors.lastname: Cheng
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
authors.forename: Xinrong
authors.initials: X
authors.lastname: Liu
authors.forename: Shuping
authors.initials: S
authors.lastname: Dou
authors.forename: Guozheng
authors.initials: G
authors.lastname: Liu
authors.forename: Minmin
authors.initials: M
authors.lastname: Liang
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Hnatowich
authors.forename: Mary
authors.initials: M
authors.lastname: Rusckowski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000000981:diagnostic imaging / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism* / Q000493:pharmacokinetics; D011485:Protein Binding; D013667:Technetium; D014018:Tissue Distribution; D015899:Tomography, Emission-Computed, Single-Photon; D023041:Xenograft Model Antitumor Assays
chemical_list: D000951:Antigens, Neoplasm; D006023:Glycoproteins; D019151:Peptide Library; D010455:Peptides; C047052:tumor-associated antigen 72; D013667:Technetium