Figure 1.

Engineering of nanobodies (Nbs) recognizing CCR7 by bimolecular fluorescence complementation (BiFC). Structure and sequence of the conformation-specific Nb80, which recognizes an isoproterenol-activated conformation of the β2-adrenergic receptor (β₂AR), was used to specifically randomize complementarity determining region (CDR)1 and CDR3 to generate a new Nb library. The newly generated Nb library was fused to the N-terminal part of split-YFP (YFP1, Y1) in order to identify Nbs that recognize CCR7 fused to split-YFP2 (Y2) by BiFC. (a) Schematic illustration of BiFC between Nb80-YFP1 and β₂AR-YFP2. Nb80 recognizes and binds to agonist activated β₂AR. Thereby, the two split-YFP fragments will reconstitute to form native YFP. (b) Flow cytometric analysis of HEK293 cells transiently expressing either Nb80-YFP1 (dotted brown line), β₂AR-YFP2 (dotted green line), or CCR7-YFP2 (dotted red line) alone. Untransfected, control cells are shown in grey. (c) Flow cytometric analysis showing BiFC in HEK293 cells transiently co-expressing Nb80-YFP1 and β₂AR-YFP2 (green line) as proof of concept or Nb80-YFP1 and CCR7-YFP2 (red line) as control. Before measuring YFP fluorescence, cells were stimulated with isoproterenol (10 µM) or CCL19 (0.5 µg/mL), respectively. (d) Negative screening of Nb library to remove β₂AR-recognizing Nbs. HEK293 cells were transiently transfected with the newly generated Nb library fused to split-YFP1 (Nb-lib-Y1) and β₂AR fused to split-YFP2. After isoproterenol stimulation (10 µM), BiFC-negative cells were FACS sorted to enrich the Nb library for Nbs that do not interact with β₂AR anymore. Sorted cell fraction is indicated by the black line. Afterwards, plasmids coding for the Nb library were isolated. (e) BiFC of remaining Nb library-YFP1 and CCR7-YFP2. The Nb library-YFP1 was transiently expressed in HEK293 cells stably expressing CCR7-YFP2 and cells were stimulated with CCL19 (0.5 µg/mL). BiFC-positive cells, indicated by the black line, were FACS sorted.