Journal of Molecular Evolution

, Volume 81, Issue 5–6, pp 210–217 | Cite as

A Fluorescent Imaging Probe Based on a Macrocyclic Scaffold That Binds to Cellular EpCAM

  • Kazuhiro Iwasaki
  • Yuki Goto
  • Takayuki Katoh
  • Taro Yamashita
  • Shuichi Kaneko
  • Hiroaki SugaEmail author
Original Article


Here, we report a fluorescent probe based on a macrocyclic peptide scaffold that specifically stains EpCAM-expressing MCF7 cells. The 14-mer macrocyclic peptide binding to the extracellular domain of EpCAM with a dissociation constant in the low nM range (1.7 nM) was discovered using the random non-standard peptide-integrated discovery system. Notably, this probe containing a fluorescence tag is less than 3000 Da in total and able to visualize nearly every live cell under high cell-density conditions, which was not achieved by the conventional mAb staining method. This suggests that the molecular probe based on the compact macrocyclic scaffold has great potentials as an imaging tool for the EpCAM biomarker as well as a delivery vehicle for drug conjugates.


In vitro selection Macrocyclic peptide EpCAM Fluorescent imaging probe Cancer cell imaging 



We thank Drs. M. Ohuchi and T. Yoshitomi for supporting in the preparation of the manuscript. This work was supported by Ministry of Health, Labor and Welfare Grants-in-aid for Scientific Research programs from 2010 to 2012 to H. S., Y. T., and S. K., Grants-in-Aid for JSPS Fellows (7734) to K. I., and partly grants of Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (A) (24681047) to T. K., and Challenging Exploratory Research (15K12739) to Y. G.

Supplementary material

239_2015_9710_MOESM1_ESM.pdf (801 kb)
Supplementary material 1 (PDF 800 kb)


  1. Armstrong A, Eck SL (2003) EpCAM: a new therapeutic target for an old cancer antigen. Cancer Biol Ther 2:320–326CrossRefPubMedGoogle Scholar
  2. Baeuerle PA, Gires O (2007) EpCAM (CD326) finding its role in cancer. Br J Cancer 96:417–423. doi: 10.1038/sj.bjc.6603494 PubMedCentralCrossRefPubMedGoogle Scholar
  3. Chaudry MA, Sales K, Ruf P, Lindhofer H, Winslet MC (2007) EpCAM an immunotherapeutic target for gastrointestinal malignancy: current experience and future challenges. Br J Cancer 96:1013–1019. doi: 10.1038/sj.bjc.6603505 PubMedCentralCrossRefPubMedGoogle Scholar
  4. Cirulli V et al (1998) KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development. J Cell Biol 140:1519–1534PubMedCentralCrossRefPubMedGoogle Scholar
  5. Deonarain MP, Kousparou CA, Epenetos AA (2009) Antibodies targeting cancer stem cells: a new paradigm in immunotherapy? MAbs 1:12–25PubMedCentralCrossRefPubMedGoogle Scholar
  6. Goto Y, Ohta A, Sako Y, Yamagishi Y, Murakami H, Suga H (2008) Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem Biol 3:120–129. doi: 10.1021/cb700233t CrossRefPubMedGoogle Scholar
  7. Goto Y, Katoh T, Suga H (2011) Flexizymes for genetic code reprogramming. Nat Protoc 6:779–790. doi: 10.1038/nprot.2011.331 CrossRefPubMedGoogle Scholar
  8. Guillemot JC, Naspetti M, Malergue F, Montcourrier P, Galland F, Naquet P (2001) Ep-CAM transfection in thymic epithelial cell lines triggers the formation of dynamic actin-rich protrusions involved in the organization of epithelial cell layers. Histochem Cell Biol 116:371–378. doi: 10.1007/s004180100329 CrossRefPubMedGoogle Scholar
  9. Hayashi Y, Morimoto J, Suga H (2012) In vitro selection of anti-Akt2 thioether-macrocyclic peptides leading to isoform-selective inhibitors. ACS Chem Biol 7:607–613. doi: 10.1021/cb200388k CrossRefPubMedGoogle Scholar
  10. Hristodorov D et al (2014) EpCAM-selective elimination of carcinoma cells by a novel MAP-based cytolytic fusion protein. Mol Cancer Ther 13:2194–2202. doi: 10.1158/1535-7163.MCT-13-0781 CrossRefPubMedGoogle Scholar
  11. Huls GA et al (1999) A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments. Nat Biotechnol 17:276–281. doi: 10.1038/7023 CrossRefPubMedGoogle Scholar
  12. Hussain S, Pluckthun A, Allen TM, Zangemeister-Wittke U (2006) Chemosensitization of carcinoma cells using epithelial cell adhesion molecule-targeted liposomal antisense against bcl-2/bcl-xL. Mol Cancer Ther 5:3170–3180. doi: 10.1158/1535-7163.MCT-06-0412 CrossRefPubMedGoogle Scholar
  13. Ito K et al (2015) Artificial human Met agonists based on macrocycle scaffolds. Nat Commun 6:6373. doi: 10.1038/ncomms7373 PubMedCentralCrossRefPubMedGoogle Scholar
  14. Iwasaki K, Goto Y, Katoh T, Suga H (2012) Selective thioether macrocyclization of peptides having the N-terminal 2-chloroacetyl group and competing two or three cysteine residues in translation. Org Biomol Chem 10:5783–5786. doi: 10.1039/c2ob25306b CrossRefPubMedGoogle Scholar
  15. Jung YK, Woo MA, Soh HT, Park HG (2014) Aptamer-based cell imaging reagents capable of fluorescence switching. Chem Commun (Camb) 50:12329–12332. doi: 10.1039/c4cc03888f CrossRefGoogle Scholar
  16. Kodan A et al (2014) Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog. Proc Natl Acad Sci USA 111:4049–4054. doi: 10.1073/pnas.1321562111 PubMedCentralCrossRefPubMedGoogle Scholar
  17. Kratz H, Haeckel A, Michel R, Schonzart L, Hanisch U, Hamm B, Schellenberger E (2012) Straightforward thiol-mediated protein labelling with DTPA: Synthesis of a highly active 111In-annexin A5-DTPA tracer. EJNMMI Res 2:17. doi: 10.1186/2191-219X-2-17 PubMedCentralCrossRefPubMedGoogle Scholar
  18. Kwiatkowska-Borowczyk EP, Gabka-Buszek A, Jankowski J, Mackiewicz A (2015) Immunotargeting of cancer stem cells. Contemp Oncol (Pozn) 19:A52–A59. doi: 10.5114/wo.2014.47129 Google Scholar
  19. Ladwein M et al (2005) The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7. Exp Cell Res 309:345–357. doi: 10.1016/j.yexcr.2005.06.013 CrossRefPubMedGoogle Scholar
  20. Linke R, Klein A, Seimetz D (2010) Catumaxomab: clinical development and future directions. MAbs 2:129–136PubMedCentralCrossRefPubMedGoogle Scholar
  21. Litvinov SV et al (1997) Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins. J Cell Biol 139:1337–1348PubMedCentralCrossRefPubMedGoogle Scholar
  22. Maetzel D et al (2009) Nuclear signalling by tumour-associated antigen EpCAM. Nat Cell Biol 11:162–171. doi: 10.1038/ncb1824 CrossRefPubMedGoogle Scholar
  23. Martin-Killias P, Stefan N, Rothschild S, Pluckthun A, Zangemeister-Wittke U (2011) A novel fusion toxin derived from an EpCAM-specific designed ankyrin repeat protein has potent antitumor activity. Clin Cancer Res 17:100–110. doi: 10.1158/1078-0432.CCR-10-1303 CrossRefPubMedGoogle Scholar
  24. Morimoto J, Hayashi Y, Suga H (2012) Discovery of macrocyclic peptides armed with a mechanism-based warhead: isoform-selective inhibition of human deacetylase SIRT2. Angew Chem Int Ed Engl 51:3423–3427. doi: 10.1002/anie.201108118 CrossRefPubMedGoogle Scholar
  25. Morioka T, Loik ND, Hipolito CJ, Goto Y, Suga H (2015) Selection-based discovery of macrocyclic peptides for the next generation therapeutics. Curr Opin Chem Biol 26:34–41. doi: 10.1016/j.cbpa.2015.01.023 CrossRefPubMedGoogle Scholar
  26. Munz M, Kieu C, Mack B, Schmitt B, Zeidler R, Gires O (2004) The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene 23:5748–5758. doi: 10.1038/sj.onc.1207610 CrossRefPubMedGoogle Scholar
  27. Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H (1997) In vitro virus: bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett 414:405–408CrossRefPubMedGoogle Scholar
  28. Nochi T et al (2004) Biological role of Ep-CAM in the physical interaction between epithelial cells and lymphocytes in intestinal epithelium. Clin Immunol 113:326–339. doi: 10.1016/j.clim.2004.08.013 CrossRefPubMedGoogle Scholar
  29. Osta WA et al (2004) EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res 64:5818–5824. doi: 10.1158/0008-5472.CAN-04-0754 CrossRefPubMedGoogle Scholar
  30. Passioura T, Katoh T, Goto Y, Suga H (2014) Selection-based discovery of druglike macrocyclic peptides. Annu Rev Biochem 83:727–752. doi: 10.1146/annurev-biochem-060713-035456 CrossRefPubMedGoogle Scholar
  31. Patriarca C, Macchi RM, Marschner AK, Mellstedt H (2012) Epithelial cell adhesion molecule expression (CD326) in cancer: a short review. Cancer Treat Rev 38:68–75. doi: 10.1016/j.ctrv.2011.04.002 CrossRefPubMedGoogle Scholar
  32. Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci USA 94:12297–12302PubMedCentralCrossRefPubMedGoogle Scholar
  33. Shaunak S et al (2006) Site-specific PEGylation of native disulfide bonds in therapeutic proteins. Nat Chem Biol 2:312–313. doi: 10.1038/nchembio786 CrossRefPubMedGoogle Scholar
  34. Song Y et al (2013) Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture. Anal Chem 85:4141–4149. doi: 10.1021/ac400366b CrossRefPubMedGoogle Scholar
  35. Spizzo G et al (2004) High Ep-CAM expression is associated with poor prognosis in node-positive breast cancer. Breast Cancer Res Treat 86:207–213. doi: 10.1023/B:BREA.0000036787.59816.01 CrossRefPubMedGoogle Scholar
  36. Spizzo G et al (2006) Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol Oncol 103:483–488. doi: 10.1016/j.ygyno.2006.03.035 CrossRefPubMedGoogle Scholar
  37. Stefan N, Martin-Killias P, Wyss-Stoeckle S, Honegger A, Zangemeister-Wittke U, Pluckthun A (2011) DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency. J Mol Biol 413:826–843. doi: 10.1016/j.jmb.2011.09.016 CrossRefPubMedGoogle Scholar
  38. Subramanian N, Sreemanthula JB, Balaji B, Kanwar JR, Biswas J, Krishnakumar S (2014) A strain-promoted alkyne-azide cycloaddition (SPAAC) reaction of a novel EpCAM aptamer-fluorescent conjugate for imaging of cancer cells. Chem Commun (Camb) 50:11810–11813. doi: 10.1039/c4cc02996h CrossRefGoogle Scholar
  39. Tanaka Y et al (2013) Structural basis for the drug extrusion mechanism by a MATE multidrug transporter. Nature 496:247–251. doi: 10.1038/nature12014 CrossRefPubMedGoogle Scholar
  40. Trzpis M, McLaughlin PM, de Leij LM, Harmsen MC (2007) Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule. Am J Pathol 171:386–395. doi: 10.2353/ajpath.2007.070152 PubMedCentralCrossRefPubMedGoogle Scholar
  41. Went P, Dirnhofer S, Salvisberg T, Amin MB, Lim SD, Diener PA, Moch H (2005) Expression of epithelial cell adhesion molecule (EpCam) in renal epithelial tumors. Am J Surg Pathol 29:83–88CrossRefPubMedGoogle Scholar
  42. Went P et al (2006) Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers. Br J Cancer 94:128–135. doi: 10.1038/sj.bjc.6602924 PubMedCentralCrossRefPubMedGoogle Scholar
  43. Yamagishi Y, Shoji I, Miyagawa S, Kawakami T, Katoh T, Goto Y, Suga H (2011) Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. Chem Biol 18:1562–1570. doi: 10.1016/j.chembiol.2011.09.013 CrossRefPubMedGoogle Scholar
  44. Zielonka S et al (2014) Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation. J Biotechnol 191:236–245. doi: 10.1016/j.jbiotec.2014.04.023 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Kazuhiro Iwasaki
    • 1
  • Yuki Goto
    • 2
  • Takayuki Katoh
    • 2
  • Taro Yamashita
    • 3
  • Shuichi Kaneko
    • 3
  • Hiroaki Suga
    • 2
    • 4
    Email author
  1. 1.Department of Chemistry and Biotechnology, Graduate School of EngineeringThe University of TokyoTokyoJapan
  2. 2.Department of Chemistry, Graduate School of ScienceThe University of TokyoTokyoJapan
  3. 3.Disease Control and Homeostasis, Graduate School of Medical SciencesKanazawa UniversityKanazawaJapan
  4. 4.JST, CRESTTokyoJapan

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