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

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.

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant.