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    Comparing two TAG-72 binding peptides previously identified by phage display as potential imaging agents

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    Authors
    Chen, Ling
    Wang, Yi
    Cheng, Dengfeng
    Dou, Shuping
    Liu, Xinrong
    Liu, Guozheng
    Hnatowich, Donald J.
    Rusckowski, Mary
    UMass Chan Affiliations
    Department of Radiology, Division of Nuclear Medicine
    Document Type
    Journal Article
    Publication Date
    2011-10-01
    Keywords
    Amino Acid Sequence
    Animals
    Antigens, Neoplasm
    Biotinylation
    Gene Expression Regulation, Neoplastic
    Glycoproteins
    HT29 Cells
    Humans
    Mice
    Molecular Sequence Data
    Neoplasms
    Organotechnetium Compounds
    *Peptide Library
    Peptides
    phage display
    TAG-72
    tumor imaging
    peptide biomarker
    Amino Acids, Peptides, and Proteins
    Medicinal and Pharmaceutical Chemistry
    Neoplasms
    Oncology
    Radiology
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    Link to Full Text
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164986/pdf/nihms-305282.pdf
    Abstract
    AIM: To evaluate the targeting property in vitro and in vivo of two tumor-associated glycoprotein 72 (TAG-72) binding peptides, previously identified in this laboratory by phage selection using different elution conditions. MATERIALS AND METHODS: The peptides GGVSCMQTSPVCENNL (A2-6) and NPGTCKDKWEICLLNGG (A3-10) were radiolabeled with technetium-99m ((99m)Tc) using N-hydroxysuccinimidyl-S-acetyl-mercaptoacetyltriglycine (NHS-MAG(3)) as a chelator or were biotinylated. The specificity of the two peptides for the TAG-72 positive LS-174T cancer cells was demonstrated in vitro both by flow cytometry analysis using the biotinylated peptides and by competitive binding using the (99m)Tc-labeled peptides. The in-vivo biodistributions of the peptides were evaluated in TAG-72 positive LS-174T tumor-bearing mice by small-animal single photon emission computed tomography/computed tomography imaging. RESULTS: As evidence of specific binding, both peptides showed a significant increase in percentage binding with increasing peptide concentration by flow cytometry analysis to LS-174T cells, but not to TAG-72 negative HT-29 cells. The (99m)Tc-labeled A2-6 peptide bound LS-174T cells with an inhibition constant at 50% of 46.5 nmol/l compared with 420 nmol/l for the A3-10 peptide. In mice, accumulation of both peptides was highest in kidneys and gallbladder. Tumors were clearly visible by single photon emission computed tomography imaging for both (99m)Tc-labeled peptides through 60 min, although the tumor accumulation was higher for the A3-10 peptide. CONCLUSION: The A3-10 peptide, with lower, yet reasonable binding affinity compared with the A2-6 peptide, showed sufficiently favorable specific binding and tumor accumulation to be considered further as a potential imaging agent for TAG-72 positive cancers.
    Source
    Nucl Med Commun. 2011 Oct;32(10):920-4. doi: 10.1097/MNM.0b013e328348fc64. Link to article on publisher's site
    DOI
    10.1097/MNM.0b013e328348fc64
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/48330
    PubMed ID
    21876403
    Related Resources
    Link to Article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1097/MNM.0b013e328348fc64
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    • Thumbnail

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

      Xiao, Nan; Cheng, Dengfeng; Wang, Yi; Chen, Ling; Liu, Xinrong; Dou, Shuping; Liu, Guozheng; Liang, Min Min; Hnatowich, Donald J.; Rusckowski, Mary (2011-01-01)
      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.
    • Thumbnail

      A new TAG-72 cancer marker peptide identified by phage display

      Chen, Ling; Wang, Yi; Liu, Xinrong; Dou, Shuping; Liu, Guozheng; Hnatowich, Donald J.; Rusckowski, Mary (2008-12-08)
      Radiolabeled peptides as markers of cancer targets have demonstrated their value in diagnostic imaging and radiotherapy. The 16 mer f88-4/Cys6 phage display library was applied to affinity purified TAG-72 and three consensus peptides were identified: VHHSCTKLTHCCQNWH (A2-13), GGVSCMQTSPVCENNL (A2-6) and TKRDCSAQNYGCQKAI (A2-11). The A2-13 and A2-6 phages showed the highest percent binding to LS-174T cells by flow cytometry and were 3-fold higher than a control phage, while fluorescence microscopy showed that both A2-6 and A2-13 phages bound to the LS-174T cell membrane. However, only the A2-6 phage demonstrated specificity by low binding to the TAG-72 negative cell HT-29. Furthermore, the synthesized free A2-6 peptide demonstrated specific binding to LS-174T cells by flow cytometry and by immunohistochemical staining of xenograft tumor compared to normal colon. These data indicate that the A2-6 peptide is specific for the TAG-72 cancer target.
    • Thumbnail

      Soluble beta-amyloid1-40 induces NMDA-dependent degradation of postsynaptic density-95 at glutamatergic synapses

      Roselli, F.; Tirard, M.; Lu, J.; Hutzler, P.; Lamberti, P.; Livrea, P.; Morabito, Maria A.; Almeida, O.F.X. (2005-12-02)
      Amyloid-beta (Abeta) has been implicated in memory loss and disruption of synaptic plasticity observed in early-stage Alzheimer's disease. Recently, it has been shown that soluble Abeta oligomers target synapses in cultured rat hippocampal neurons, suggesting a direct role of Abeta in the regulation of synaptic structure and function. Postsynaptic density-95 (PSD-95) is a postsynaptic scaffolding protein that plays a critical role in synaptic plasticity and the stabilization of AMPA (AMPARs) and NMDA (NMDARs) receptors at synapses. Here, we show that exposure of cultured cortical neurons to soluble oligomers of Abeta(1-40) reduces PSD-95 protein levels in a dose- and time-dependent manner and that the Abeta1(1-40)-dependent decrease in PSD-95 requires NMDAR activity. We also show that the decrease in PSD-95 requires cyclin-dependent kinase 5 activity and involves the proteasome pathway. Immunostaining analysis of cortical cultured neurons revealed that Abeta treatment induces concomitant decreases in PSD-95 at synapses and in the surface expression of the AMPAR glutamate receptor subunit 2. Together, these data suggest a novel pathway by which Abeta triggers synaptic dysfunction, namely, by altering the molecular composition of glutamatergic synapses.
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