Cy5.5-labeled Affibody molecule for near-infrared fluorescent optical imaging of epidermal growth factor receptor positive tumors. J Biomed Opt 15(3):036007

Stanford University, Stanford Cancer Center, Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, 1201 Welch Road, Lucas Expansion, P095, Stanford, California 94305-5344, USA.
Journal of Biomedical Optics (Impact Factor: 2.86). 05/2010; 15(3):036007. DOI: 10.1117/1.3432738
Source: PubMed


Affibody protein is an engineered protein scaffold with a three-helical bundle structure. Affibody molecules of small size (7 kD) have great potential for targeting overexpressed cancer biomarkers in vivo. To develop an Affibody-based molecular probe for in vivo optical imaging of epidermal growth factor receptor (EGFR) positive tumors, an anti-EGFR Affibody molecule, Ac-Cys-Z(EGFR:1907) (7 kD), is site-specifically conjugated with a near-IR fluorescence dye, Cy5.5-mono-maleimide. Using fluorescent microscopy, the binding specificity of the probe Cy5.5-Z(EGFR:1907) is checked by a high-EGFR-expressing A431 cell and low-EGFR-expressing MCF7 cells. The binding affinity of Cy5.5-Z(EGFR:1907) (K(D)) to EGFR is 43.6+/-8.4 nM, as determined by flow cytometry. For an in vivo imaging study, the probe shows fast tumor targeting and good tumor contrast as early as 0.5 h postinjection (p.i.) for A431 tumors, while MCF7 tumors are barely visible. An ex vivo imaging study also demonstrates that Cy5.5-Z(EGFR:1907) has high tumor, liver, and kidney uptakes at 24 h p.i.. In conclusion, Cy5.5-Z(EGFR:1907) shows good affinity and high specificity to the EGFR. There is rapid achievement of good tumor-to-normal-tissue contrasts of Cy5.5-Z(EGFR:1907), thus demonstrating its potential for EGFR-targeted molecular imaging of cancers.

16 Reads
  • Source
    • "Probes and blocking agents (10 mM/6 mL) were injected into the knee joints through the patellar ligament when joint was flexed to 90 (n ¼ 4 for each group). In vivo fluorescence imaging was performed using an IVIS 200 system and a Cy5 filter set (excitation 615e655 nm; emission 695e770 nm) for Cy5-DPA-Zn and Cy5-DPA; while IVIS spectrum system with a Cy7 filter set (695e735 nm; emission 750e850 nm) was used for Cy7-DPA-Zn and Cy7-DPA, following previous protocol [27]. The animals were anesthetized and imaged at 0.5, 1, 2, 4, 12, 24, 48, 72 h post-injection of the probes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Articular cartilage is the hydrated tissue that lines the ends of long bones in load bearing joints and provides joints with a smooth, nearly frictionless gliding surface. However, the deterioration of articular cartilage occurs in the early stages of osteoarthritis (OA) and is clinically and radiographically silent. Here two cationic near infrared fluorescent (NIRF) dipicolylamine (DPA) probes, Cy5-DPA-Zn and Cy7-DPA-Zn, were prepared for cartilage degeneration imaging and OA early detection through binding to the anionic glycosaminoglycans (GAGs). The feasibility of NIRF dye labeled DPA-Zn probes for cartilage degeneration imaging was examined ex vivo and in vivo. The ex vivo studies showed that Cy5-DPA-Zn and Cy7-DPA-Zn not only showed the high uptake and electrostatic attractive binding to cartilage, but also sensitively reflected the change of GAGs contents. In vivo imaging study further indicated that Cy5-DPA-Zn demonstrated higher uptake and retention in young mice (high GAGs) than old mice (low GAGs) when administrated via local injection in mouse knee joints. More importantly, Cy5-DPA-Zn showed dramatic higher signals in sham joint (high GAGs) than OA side (low GAGs), through sensitive reflecting the change of GAGs in the surgical induced OA models. In summary, Cy5-DPA-Zn provides promising visual detection for early cartilage pathological degeneration in living subjects.
    Biomaterials 06/2014; 35(26). DOI:10.1016/j.biomaterials.2014.05.042 · 8.56 Impact Factor
  • Source
    • "Several classes of scaffold protein-based radiolabeled probes have a high potential for in vivo molecular imaging, including affibody molecules, cystine knot peptides, and nanobodies.52 Several selected therapeutic targets have been developed that affibody molecules exhibit high-affinity binding to, such as HER2, EGFR, insulin-like growth factor 1, and platelet-derived growth factor receptor B.53–55 Anti-EGFR affibody-based molecular probes, 64Cu-DOTA-Cys-ZEGFR:1907 and Cy5.5-Cys-ZEGFR:1907, were successfully prepared and found to show rapid tumor targeting ability and good tumor imaging contrast even at 1 hour after injection in the EGFR-expressing A431 tumor xenograft model.56,57 In addition, radiolabeled affibody molecules have been used to monitor the degradation of HER2 in response to inhibition of HSP90,58,59 showing that they might be used not only for patient stratification, but also for therapy monitoring. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Targeted therapy is becoming an increasingly important component in the treatment of cancer. How to accurately monitor targeted therapy has been crucial in clinical practice. The traditional approach to monitor treatment through imaging has relied on assessing the change of tumor size by refined World Health Organization criteria, or more recently, by the Response Evaluation Criteria in Solid Tumors. However, these criteria, which are based on the change of tumor size, show some limitations for evaluating targeted therapy. Currently, genetic alterations are identified with prognostic as well as predictive potential concerning the use of molecularly targeted drugs. Conversely, considering the limitations of invasiveness and the issue of expression heterogeneity, molecular imaging is better able to assay in vivo biologic processes noninvasively and quantitatively, and has been a particularly attractive tool for monitoring treatment in clinical cancer practice. This review focuses on the applications of different kinds of molecular imaging including positron emission tomography-, magnetic resonance imaging-, ultrasonography-, and computed tomography-based imaging strategies on monitoring targeted therapy. In addition, the key challenges of molecular imaging are addressed to successfully translate these promising techniques in the future.
    International Journal of Nanomedicine 09/2013; 8:3703-3713. DOI:10.2147/IJN.S51264 · 4.38 Impact Factor
  • Source
    • "In vivo imaging with 64 Cu-or Cy5.5-labeled Z EGFR:1907 showed fast tumor (A431) targeting and good tumor-to-normal tissue contrast. Both agents accumulated at a high level in tumor, liver and kidney as revealed by biodistribution study (Miao et al., 2010, 2010). A dimeric form of EGFR-specific affibody (13.7 kDa) was labeled with IRDye 800CW (named as Eaff800), and used to image A431 xenograft tumors. "

    Molecular Imaging, 03/2012; , ISBN: 978-953-51-0359-2
Show more

Similar Publications