Development of a generalisable tryptophan-optimised quenchbody biosensor based on a synthetic nanobody library

Quenchbodies are antibodies labelled with a fluorophore that increases in fluorescence intensity upon antigen binding, which makes them promising candidates for the development of diagnostic or other immunoassays requiring antigen quantification. Quenchbodies based on camelid nanobodies are particularly attractive for rapid development of immunoassays against molecular antigens of interest, due to their small size, ease of expression, high stability, rapid evolvability, and amenability to protein engineering. However, current nanobody-based quenchbodies display only modest fluorescence increases when binding to protein antigens (≤ 1.1-1.4-fold), with few examples reaching > 2-fold. Here we show that tryptophan residues in the nanobody CDRs are critical to fluorescence antigen detection, which subsequently informed the development of a synthetic convex-binding nanobody library that was used for the in vitro production of novel quenchbodies against human inflammatory cytokine interleukin 6 (IL6). To inform sequence development for the nanobody library, initial in silico modelling and biochemical analyses showed that existing nanobodies for maltose-binding protein (MBP) and lysozyme could be converted into quenchbodies (Qb-MBP and Qb-Lys), with fluorescence fold-increases of 1.5 and 1.3, respectively, when recognising their cognate antigen. Rational mutational substitution of tryptophans into the CDR-region of the quenchbodies resulted in fluorescence fold-increases of 1.9 and 1.6 for Qb-MBP (Y59W/Y114W) and Qb-Lys (Y110W), respectively, supporting the importance of CDR-based tryptophans in the nanobody quenchbody mechanism. A synthetic nanobody library enriched with CDR-based tryptophans was subsequently created, and biochemical analyses revealed quenchbodies evolved against interleukin-6 (IL6) with fluorescence fold-increases of 1.5-2.4 (EC50 = 20-1,113 nM binding affinity). The ease and speed by which nanobody-based quenchbodies can be discovered using this completely in vitro selection strategy based on a single synthetic library, makes this a very attractive approach to develop immunoassays for detection of a wide range of molecular targets, including proteins.