title: Evaluation of four affibody-based near-infrared fluorescent probes for optical imaging of epidermal growth factor receptor positive tumors.
abstract: The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, ZEGFR:1907, has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors.
  
  
  METHODS
  The Affibody analogue, Ac-Cys-ZEGFR:1907, was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probes-Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, SR680-ZEGFR:1907, and 800CW-ZEGFR:1907. The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (KD) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors.
  
  
  RESULTS
  The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 possessed high binding affinity in low nanomolar range (43.6 ± 8.4 and 28.3 ± 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated that the four probes had high tumor uptakes. Particularly, Cy5.5-ZEGFR:1907 and Alex680-ZEGFR:1907 displayed higher tumor-to-normal tissue ratios than those of the other two probes.
  
  
  CONCLUSIONS
  This work demonstrates that Affibody proteins can be modified with different NIR fluorescent dyes and used for imaging of EGFR expressing tumors. Different NIR fluorescent dyes have variable impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development. The probes developed are promising for further tumor imaging applications and clinical translation. Particularly, Alex680-ZEGFR:1907 and Cy5.5-ZEGFR:1907 are excellent candidates as EGFR-targeted probes for optical imaging.
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology, and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University , Stanford, California, 94305-5344.
authors.forename: Shibo
authors.initials: S
authors.lastname: Qi
authors.forename: Zheng
authors.initials: Z
authors.lastname: Miao
authors.forename: Hongguang
authors.initials: H
authors.lastname: Liu
authors.forename: Yingding
authors.initials: Y
authors.lastname: Xu
authors.forename: Yaqing
authors.initials: Y
authors.lastname: Feng
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cheng
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000032:analysis*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D009369:Neoplasms / Q000175:diagnosis*; D061848:Optical Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: C569382:(Z(EGFR)-1907)2 affibody; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors