Inhalable Nanofitin demonstrates high neutralization of SARS-CoV-2 virus via direct application in respiratory tract.

Nanofitins are small and hyperthermostable alternative protein scaffolds that display physicochemical properties making them suitable for the development of topical therapeutics, notably for the treatment of pulmonary infectious diseases. Local administration of biologics to the lungs involves a particularly stressful step of nebulization that is poorly tolerated by most antibodies, which limits their application by this delivery route. During the COVID-19 pandemic, we generated anti-SARS-CoV-2 monomeric Nanofitins of high specificity for the spike protein. Hit Nanofitin candidates were identified based on their binding properties with punctual spike mutants and assembled into a linear multimeric construction constituting of four different Nanofitins, allowing the generation of a highly potent anti-SARS-CoV-2 molecule. The therapeutic efficacy of the multimeric assembly was demonstrated both in in vitro and in vivo models. Interestingly, the neutralization mechanism of the multimeric construction seems to involve a particular conformation switch of the spike trimer. In addition, we reported the stability and the conserved activity of the tetrameric construction after nebulization. This advantageous developability feature for pulmonary administration associated with the ease of assembly, as well as the fast generation process position the Nanofitin technology as a potential therapeutic solution for emerging infectious diseases.

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.