Optimization of single-domain antibodies functionalization for lung administration

The use of therapeutic proteins for lung diseases remains challenging, especially in terms of drug delivery and pharmacokinetic. In our project, we aimed to investigate the use of single-domain antibodies (Nanobodies) as potential tools for the specific targeting of lung antigens. We selected four potential lung targets (i.e. serum albumin, protein A of lung surfactant, collagen type III and the SEA domain of mucin) and their cognate nanobodies (Nbs) have been synthesized in E. coli by our partner from University of Liège. We first developed and optimized the labeling of Nbs with fluorescent probes, to follow their biodistribution in vivo. In a second part we developed an ELISA assay to dose the amount of Nbs in mice. Reduction of dimers followed by the conjugation with VT-S-750 maleimide fluorescent probe showed an absorption on agarose beads bearing tris(2-carboxyethyl)phosphine (TCEP) and a complete quenching of fluorescence when using free TCEP. Reduction with 2-Mercaptoethylamine-HCl (2-MEA) however allowed a better conservation of activity of our labeled-Nbs compared to TCEP. ELISA assays performed on Phe-Nbs allowed the recovery of only 12 to 15% of the total amount of Nbs in mice at basal time after lung administration. We succeeded to develop a good and reproducible method for the fluorescent labelling of Cterm-Nbs with an acceptable loss of affinity. However, we failed to obtain a complete purification using size-exclusion chromatography which suggests that other separation methods or columns should be used. The ELISA was unsuccessful, so other prospects are currently explored to perform pharmacokinetic studies in mice.