Nanofitin as a New Molecular-Imaging Agent for the Diagnosis of Epidermal Growth Factor Receptor Over-Expressing Tumors

Bioconjug Chem. 2017 Sep 20;28(9):2361-2371. doi: 10.1021/acs.bioconjchem.7b00374. Epub 2017 Sep 12.


Epidermal growth-factor receptor (EGFR) is involved in cell growth and proliferation and is over-expressed in malignant tissues. Although anti-EGFR-based immunotherapy became a standard of care for patients with EGFR-positive tumors, this strategy of addressing cancer tumors by targeting EGFR with monoclonal antibodies is less-developed for patient diagnostic and monitoring. Indeed, antibodies exhibit a slow blood clearance, which is detrimental for positron emission tomography (PET) imaging. New molecular probes are proposed to overcome such limitations for patient monitoring, making use of low-molecular-weight protein scaffolds as alternatives to antibodies, such as Nanofitins with better pharmacokinetic profiles. Anti-EGFR Nanofitin B10 was reformatted by genetic engineering to exhibit a unique cysteine moiety at its C-terminus, which allows the development of a fast and site-specific radiolabeling procedure with 18F-4-fluorobenzamido-N-ethylamino-maleimide (18F-FBEM). The in vivo tumor targeting and imaging profile of the anti-EGFR Cys-B10 Nanofitin was investigated in a double-tumor xenograft model by static small-animal PET at 2 h after tail-vein injection of the radiolabeled Nanofitin 18F-FBEM-Cys-B10. The image showed that the EGFR-positive tumor (A431) is clearly delineated in comparison to the EGFR-negative tumor (H520) with a significant tumor-to-background contrast. 18F-FBEM-Cys-B10 demonstrated a significantly higher retention in A431 tumors than in H520 tumors at 2.5 h post-injection with a A431-to-H520 uptake ratio of 2.53 ± 0.18 and a tumor-to-blood ratio of 4.55 ± 0.63. This study provides the first report of Nanofitin scaffold used as a targeted PET radiotracer for in vivo imaging of EGFR-positive tumor, with the anti-EGFR B10 Nanofitin used as proof-of-concept. The fast generation of specific Nanofitins via a fully in vitro selection process, together with the excellent imaging features of the Nanofitin scaffold, could facilitate the development of valuable PET-based companion diagnostics.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / pharmacokinetics
  • Cell Line, Tumor
  • Cysteine / chemistry*
  • Cysteine / pharmacokinetics
  • ErbB Receptors / analysis*
  • Female
  • Humans
  • Maleimides / chemistry*
  • Maleimides / pharmacokinetics
  • Mice, Inbred BALB C
  • Mice, Nude
  • Neoplasms / diagnosis*
  • Positron-Emission Tomography / methods*


  • Antibodies, Monoclonal
  • Maleimides
  • N-(2-(4-18F-fluorobenzamido)ethyl)maleimide
  • ErbB Receptors
  • Cysteine