Engineering of Nanobodies Recognizing the Human Chemokine Receptor CCR7

The chemokine receptor CCR7 plays a pivotal role in health and disease. In particular, CCR7 controls homing of antigen-bearing dendritic cells and T cells to lymph nodes, where adaptive immune responses are initiated. However, CCR7 also guides T cells to inflamed synovium and thereby contributes to rheumatoid arthritis and promotes cancer cell migration and metastasis formation. Nanobodies have recently emerged as versatile tools to study G-protein-coupled receptor functions and are being tested in diagnostics and therapeutics. In this study, we designed a strategy to engineer novel nanobodies recognizing human CCR7. We generated a nanobody library based on a solved crystal structure of the nanobody Nb80 recognizing the β2-adrenergic receptor (β2AR) and by specifically randomizing two segments within complementarity determining region 1 (CDR1) and CDR3 of Nb80 known to interact with β2AR. We fused the nanobody library to one half of split-YFP in order to identify individual nanobody clones interacting with CCR7 fused to the other half of split-YFP using bimolecular fluorescence complementation. We present three novel nanobodies, termed Nb1, Nb5, and Nb38, that recognize human CCR7 without interfering with G-protein-coupling and downstream signaling. Moreover, we were able to follow CCR7 trafficking upon CCL19 triggering using Nb1, Nb5, and Nb38.


Introduction
The system of chemokine receptors and their ligands, the chemokines, is crucial for guiding cell migration in development, health, and disease. Chemokines are small, secreted chemotactic cytokines that play a major role in tightly coordinating the migration and positioning of immune cells, thereby essentially contributing to both development of the immune system and regulation of innate and adaptive immune responses [1][2][3]. However, chemokines also orchestrate cancer cell dissemination and metastasis formation [4]. As a consequence, chemokines and their receptors have emerged as therapeutic targets, particularly in immune and inflammatory disorders as well as in cancer [5].
The chemokine receptor CCR7, together with its ligands, CCL19 and CCL21, orchestrates the migration of antigen-loaded dendritic cells (DCs) and lymphocytes to lymphoid organs to launch specific immune responses against invading pathogens [6,7]. CCL21 is constantly produced by lymphatic endothelial cells in peripheral tissues forming immobilized chemokine gradients from the interstitium towards lymphatic vessels [8]. Upon pathogen encountering in peripheral tissues,

Engineering of Nbs Recognizing Human CCR7
Nbs recently emerged as attractive and versatile tools for research purposes [21,23,24,26] and are being tested in diagnostics and therapeutics [27]. Nbs possess many advantages over conventional antibodies, including their small size, thermal stability and unique three-dimensional structure, which allows binding to cavities or clefts on the surface of proteins that are mostly inaccessible to conventional antibodies. Most prominently, nanobody Nb80 is considered as conformation-specific Nb recognizing active β 2 AR [21]. Crystallography revealed that Nb80 especially binds with the third complementarity determining region (CDR3) to the cytoplasmic end of β 2 AR and protrudes into the core of the receptor [21]. More precisely, an eight amino acid long sequence of CDR3 penetrates into a hydrophobic pocket established by amino acids of the receptor's transmembrane helices 3, 5, 6, and 7. In addition, a four amino acid sequence of the Nb80 s CDR1 stabilizes the interaction with regions of helices 5 and 6 of the GPCR [21]. The interaction of Nb80 with β 2 AR stabilizes a conformational state that highly resembles the active state of isoproterenol-stimulated receptor in complex with the G-protein [21,26]. To avoid the need of immunizing Llama and thus the requirement of large amounts of purified, reconstituted and activated CCR7, we intended to engineer Nbs recognizing CCR7 by taking advantage of the conserved structural architecture of GPCRs and the detailed structural information, which is available for Nb80. More precisely, we applied synthetic randomization to CDR1 and CDR3 of Nb80 with the aim to lose the affinity of new Nbs for β 2 AR while gaining specificity to CCR7, combined with the powerful and highly sensitive BiFC approach [16,28]. To this end, we cloned the newly generated Nb cDNA library in frame to the coding sequence of the N-terminal part of split-YFP1. By co-expressing the Nb library fused to YFP1 together with CCR7 fused to split-YFP2, we anticipated to identify Nbs that interact with human CCR7 by BiFC. Furthermore, we stimulated CCR7 with CCL19 hoping to also identify Nbs recognizing an active conformation of the receptor. As proof of principle for detecting Nb-GPCR interaction by BiFC, we first co-expressed Nb80-YFP1 together with β 2 AR-YFP2 in HEK293 cells and stimulated the cells with isoproterenol, a ligand of β 2 AR (Figure 1a). Notably, no BiFC was detected in HEK293 cells transfected with only Nb80-YFP1, β 2 AR-YFP2, or CCR7-YFP2 ( Figure 1b). However, we observed BiFC between Nb80-YFP1 and β 2 AR-YFP2 (Figure 1c). By co-expressing Nb80-YFP1 and CCR7-YFP2, we noted some interaction, but the YFP fluorescence intensity was much weaker compared to the one derived from the Nb80-YFP1 and β 2 AR-YFP2 BiFC (Figure 1c). This prompted us to first implement an initial negative screening, in which we transfected cells with our Nb library-YFP1 together with β 2 AR-YFP2, sorted for BiFC-negative cells ( Figure 1d) and isolated plasmids coding for the Nb library. The remaining Nb library fused to YFP1 was subsequently transfected into HEK293 cells stably expressing CCR7-YFP2, stimulated with CCL19 and BiFC-positive cells were sorted (Figure 1e). Plasmids coding for Nb-YFP1 were isolated and individual clones were co-transfected again with CCR7-YFP2. The three most promising Nb clones, referred to as Nb1, Nb5, and Nb38 (Figure 2a), were sequenced and further analyzed. As observed for Nb80 and CCR7, the CCR7-recognizing Nbs retain some basal interaction with β 2 AR (Figure 2a). This might be explained by the fact that GPCRs flicker between different conformational states and that protein-protein interactions within the BiFC system are relatively long-lived.

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. 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 (β 2 AR), 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 β 2 AR-YFP2. Nb80 recognizes and binds to agonist activated β 2 AR. 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), β 2 AR-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 β 2 AR-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 β 2 AR-recognizing Nbs. HEK293 cells were transiently transfected with the newly generated Nb library fused to split-YFP1 (Nb-lib-Y1) and β 2 AR 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 β 2 AR 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. Comparing the CDR1 and CDR3 regions of the three selected CCR7-recognizing Nb clones revealed that the two binding domains of Nb38 mainly consist of hydrophobic amino acids similar to the CDR1 and CDR3 region of Nb80 ( Figure 2b). Notably, the binding domains of Nb1 and Nb5 contain more polar amino acids, and in the case of Nb5, include charged residues.

Nb1, Nb5, and Nb38 Preferentially Recognize CCR7 While Nb80 Preferentially Interacts with β2AR
To circumvent the limitations of the BiFC system, we next established a method that allows determining more transient and dynamic protein-protein interactions. To this end, we applied a split-luciferase complementation assay, in which we co-expressed either β2AR fused to Small BiT (SmBiT) (Figure 3a) or CCR7-SmBiT ( Figure 3c) together with Nb fused to Large BiT (LgBiT) of the NanoLuc (NLuc) luciferase in HEK293 cells. The advantage of this system is that the luciferase falls apart into the split parts if the fused proteins of interest no longer interact with each other. Exploiting this split-luciferase complementation assay revealed that Nb80 predominantly interacted with activated β2AR (Figure 3b), whereas the newly engineered Nb1, Nb5, and Nb38 predominantly recognized CCR7 independent of its activation state ( Figure 3d). Here, the three most promising Nb clones are represented: Nb1, Nb5, and Nb38. BiFC between individual Nb clones and CCR7 is indicated in red. Additionally, BiFC of individual Nb-YFP1 clones and β 2 AR-YFP2 was analyzed and is depicted in green. (b) Nb1, Nb5, and Nb38 were sequenced. Protein sequences of CDR1 and CDR3 of each Nb are illustrated in comparison to Nb80. Different colors were used to highlight characteristic properties of respective amino acids (aa).
Comparing the CDR1 and CDR3 regions of the three selected CCR7-recognizing Nb clones revealed that the two binding domains of Nb38 mainly consist of hydrophobic amino acids similar to the CDR1 and CDR3 region of Nb80 ( Figure 2b). Notably, the binding domains of Nb1 and Nb5 contain more polar amino acids, and in the case of Nb5, include charged residues.

Nb1, Nb5, and Nb38 Preferentially Recognize CCR7 While Nb80 Preferentially Interacts with β 2 AR
To circumvent the limitations of the BiFC system, we next established a method that allows determining more transient and dynamic protein-protein interactions. To this end, we applied a split-luciferase complementation assay, in which we co-expressed either β 2 AR fused to Small BiT (SmBiT) (Figure 3a) or CCR7-SmBiT ( Figure 3c) together with Nb fused to Large BiT (LgBiT) of the NanoLuc (NLuc) luciferase in HEK293 cells. The advantage of this system is that the luciferase falls apart into the split parts if the fused proteins of interest no longer interact with each other. Exploiting this split-luciferase complementation assay revealed that Nb80 predominantly interacted with activated β 2 AR (Figure 3b), whereas the newly engineered Nb1, Nb5, and Nb38 predominantly recognized CCR7 independent of its activation state ( Figure 3d).

Figure 3.
Nb80 preferentially interacts with active β2AR, whereas Nb1, Nb5, and Nb38 preferentially recognize CCR7 independent of its activation state as assessed by split-luciferase complementation. (a, c) Schematic representation of the split-luciferase complementation assay. Nb-GPCR interactions are determined by reconstitution of Small BiT (SmBiT) and Large BiT (LgBiT) to functional NanoLuc (NLuc) luciferase before and after agonist stimulation and subsequent measurements of luminescence signals generated by the reconstituted luciferase. (b, d) HEK293 cells transiently co-expressing β2AR (b) or CCR7 (d) fused to SmBiT of NLuc and individual Nb clones fused to LgBiT of NLuc were incubated with coelenterazine H (5µM), the luciferase's substrate, and after 10 min, stimulated with isoproterenol (iso) (10 µM) (b) or CCL19 (1.5 µg/ml) (d). As control, we transiently co-expressed LgBiT without Nb together with either GPCR-SmBiT. Reconstituted luciferase activity between Nb80 and β2AR (b) and Nb1 and CCR7 (d), respectively, was set to 100%. Results represent each the mean values of three independent experiments including the standard error of the mean (SEM).

Nb1, Nb5, and Nb38 Barely Interfere with G-Protein-Coupling to CCR7
As Nb80 binding to agonist-activated β2AR is known to inhibit G-protein activation and consequently interferes with downstream signaling [29], we further examined the possibility of CCR7-interacting Nbs to interfere with CCR7-driven G-protein-coupling. To achieve this, we conducted a novel G-protein competition assay based on split-luciferase complementation ( Figure  4a). As proof of concept, we first tested the inhibitory capacity of Nb80 on G-protein-coupling to β2AR upon isoproterenol stimulation. In fact, Nb80 completely blocked G-protein interaction with ligand-stimulated β2AR (Figure 4b). In contrast, Nb1, Nb5, and Nb38 barely interfered with CCL19-driven G-protein-coupling to CCR7 (Figure 4c-e). . Nb80 preferentially interacts with active β 2 AR, whereas Nb1, Nb5, and Nb38 preferentially recognize CCR7 independent of its activation state as assessed by split-luciferase complementation. (a,c) Schematic representation of the split-luciferase complementation assay. Nb-GPCR interactions are determined by reconstitution of Small BiT (SmBiT) and Large BiT (LgBiT) to functional NanoLuc (NLuc) luciferase before and after agonist stimulation and subsequent measurements of luminescence signals generated by the reconstituted luciferase. (b,d) HEK293 cells transiently co-expressing β 2 AR (b) or CCR7 (d) fused to SmBiT of NLuc and individual Nb clones fused to LgBiT of NLuc were incubated with coelenterazine H (5µM), the luciferase's substrate, and after 10 min, stimulated with isoproterenol (iso) (10 µM) (b) or CCL19 (1.5 µg/mL) (d). As control, we transiently co-expressed LgBiT without Nb together with either GPCR-SmBiT. Reconstituted luciferase activity between Nb80 and β 2 AR (b) and Nb1 and CCR7 (d), respectively, was set to 100%. Results represent each the mean values of three independent experiments including the standard error of the mean (SEM).

Nb1, Nb5, and Nb38 Barely Interfere with G-Protein-Coupling to CCR7
As Nb80 binding to agonist-activated β 2 AR is known to inhibit G-protein activation and consequently interferes with downstream signaling [29], we further examined the possibility of CCR7-interacting Nbs to interfere with CCR7-driven G-protein-coupling. To achieve this, we conducted a novel G-protein competition assay based on split-luciferase complementation ( Figure 4a). As proof of concept, we first tested the inhibitory capacity of Nb80 on G-protein-coupling to β 2 AR upon isoproterenol stimulation. In fact, Nb80 completely blocked G-protein interaction with ligand-stimulated β 2 AR (Figure 4b). In contrast, Nb1, Nb5, and Nb38 barely interfered with CCL19-driven G-protein-coupling to CCR7 (Figure 4c

Nb1, Nb5, or Nb38 do not Impair CCR7-Driven Calcium Mobilization and Receptor Endocytosis
To further characterize the newly engineered CCR7-recognizing Nbs, we next determined whether the Nbs interfere with chemokine-driven calcium mobilization and receptor internalization.

Nb1, Nb5, or Nb38 do not Impair CCR7-Driven Calcium Mobilization and Receptor Endocytosis
To further characterize the newly engineered CCR7-recognizing Nbs, we next determined whether the Nbs interfere with chemokine-driven calcium mobilization and receptor internalization. Our results revealed that none of the CCR7-recognizing Nbs interfered with either CCL19-mediated mobilization of calcium ions from intracellular stores (Figure 5a) or CCL19-mediated CCR7 endocytosis (Figure 5b). These data provide clear evidence that CCR7 signaling is not compromised in the presence of the newly engineered CCR7-recognizing Nbs. Our results revealed that none of the CCR7-recognizing Nbs interfered with either CCL19-mediated mobilization of calcium ions from intracellular stores (Figure 5a) or CCL19-mediated CCR7 endocytosis (Figure 5b). These data provide clear evidence that CCR7 signaling is not compromised in the presence of the newly engineered CCR7-recognizing Nbs.

Monitoring CCL19-induced CCR7 trafficking by Nb1, Nb5, and Nb38
Finally, we assessed the newly engineered Nbs for their capacity to detect CCR7 at the plasma membrane and at endocytic vesicles upon chemokine stimulation by confocal microscopy. In line with our BiFC assays shown in Figure 1, we observed that Nb1 (Figure 6a), Nb5 ( Figure 6b) and Nb38 ( Figure 6c) interacted with CCR7 by BiFC. Notably, in the absence of ligands, BiFC between the three Nb-YFP1 clones and CCR7-YFP2 was primarily observed at the plasma membrane. Upon CCL19 stimulation, BiFC was also found at membrane ruffles and in vesicular structures. Latter was most pronounced after prolonged stimulation with chemokine.
Taken together, in the present study we designed a novel strategy to successfully engineer Nbs interacting with the chemokine receptor CCR7. We found that a selection of newly engineered Nbs recognize human CCR7 independent of its activation state and without interfering with G-protein-coupling, chemokine-mediated calcium mobilization or receptor internalization, but allow following CCR7 trafficking upon chemokine engagement in space and time.

Discussion
The chemokine receptor CCR7 plays a crucial role in guiding migration of immune cells, particularly of DCs and T cells, into secondary lymphoid organs to launch adaptive immune responses [6,7,14]. However, CCR7-mediated cell migration also contributes to inflammatory diseases, such as rheumatoid arthritis [10], or facilitates metastasis of cancer cells [11]. Moreover, misguidance of immune cells due to impaired CCR7 signaling may lead to autoimmune diseases [9]. Despite the well appreciated central role of CCR7-driven cell migration in health and disease, molecular mechanisms how CCR7 signaling controls cell migration are far from being understood. Hence, new molecular insights into how CCR7 signaling guides cell migration are highly desired. To gain such molecular insights, new tools are required to assess and monitor CCR7 signaling.
In this study, we designed a strategy to develop Nbs recognizing CCR7. Using this strategy, we successfully engineered Nbs recognizing human CCR7 by synthetic randomization of the binding domains CDR1 and CDR3 of the β 2 AR conformation-specific Nb80. In order to identify single Nb clones that interact with CCR7 out of a Nb library of more than a million clones, we exploited a high-throughput BiFC approach combined with single cell sorting. We subsequently selected individual CCR7-recognizing Nbs based on the ability to interact with CCR7 over β 2 AR by using a split-luciferase.
Nb80 was isolated and identified upon immunization of Llama with purified, agonist-bound β 2 AR that was reconstituted at high density into phospholipid vesicles [21]. To our knowledge, no one has achieved to purify CCR7 in sufficient amounts to immunize Llamas to generate CCR7-specific nanobodies, not spoken of the need to complex the purified receptor with a ligand for immunization to potentially get nanobodies reacting with active receptors. Notably, Nb80 stabilizes the active conformation of β 2 AR, which is ideal for crystallization studies. Due to its high affinity, Nb80, however, interferes with G-protein-coupling of β 2 AR and consequently dampened agonist-driven cAMP production and β-arrestin recruitment [29] (Figure 4b). In contrast, our CCR7-recognizing Nbs do not discriminate between inactive and agonist-stimulated states of the receptor and barely interfere with chemokine-mediated G-protein-coupling ( Figure 4) or downstream signaling ( Figure 5). Hence, it is unlikely that our Nbs recognizing CCR7 stabilize the receptor in a particular conformation. Moreover, as no structural information is available for CCR7, it remains to be determined how similar G-protein-coupling to CCR7 and β 2 AR is. However, it is reasonable that differences between Nb80 binding to β 2 AR and Nb1/5/38 binding to CCR7 are due to alternative biochemical properties of the polypeptide chains within CDR1 and CDR3 of individual Nb clones and the GPCR. Notably, Nb1 includes a glycine residue in CDR3, which confers high flexibility, whereas Nb5 possesses more charged amino acids in CDR3. Interestingly, the presence of proline in Nb1 gives a hint that protrusion of the long CDR3 loop into the receptor cavity might be impeded. This is consistent with the observation that Nb1 did not affect G-protein-coupling at all whereas Nb5 and Nb38 slightly reduced G-protein-coupling to CCR7. We used, in this assay, mini-Gα i (mGα i )-proteins, which functionally mimic the nucleotide-free G-protein bound to GPCR, as surrogate for heterotrimeric G-proteins since these mG-proteins are reported as excellent sensors for activation of GPCRs [30]. Since CCR7 predominantly couples to Gα i , we used the same mG-protein also for the positive control, the β 2 AR, even though it is well established that β 2 AR preferentially couples to Gα s but secondarily also couples to Gα i just with lower potency [30]. Despite this inhibitory role in β 2 AR signaling, Nb80 fused to GFP was successfully used as biosensor to identify active β 2 AR at both the plasma membrane and subsequently at endosomes upon agonist triggering [22]. Similarly, our newly generated Nbs recognized CCR7 at the plasma membrane and, upon CCL19 triggering, also at endocytic vesicles ( Figure 6).
Finally, our newly developed strategy to engineer Nbs can be used and further developed to generate additional Nbs for other GPCRs and might foster the use of such Nbs for future diagnostic and therapeutic purposes.

Reagents and Antibodies
Recombinant human CCL19 and CCL21 were purchased from PeproTech (Rocky Hill, CT, USA), ionomycin and the chemical isoproterenol hydrochloride were obtained from Sigma-Aldrich (

Synthetic Randomization of Nb80 and Construction of a Nb Library into the BiFC Vector
The β 2 AR conformation-specific Nb80 was modified by custom made synthetic randomization (Thermo Fischer Scientific) within CDR1 and CDR3. The synthetically randomized Nb library was amplified by PCR using specific primers (forward: 5 GAA GGG TAC CAA GCT TGA AAT GGT GCA G 3 ; reverse: 5 GAA GGA GCT CAT CGA TTT TGT GGT GGC 3 ). Full length fragments were gel purified und resuspended in TE-buffer revealing a total amount of 11.7 µg of amplified library. The resulting library correctness amounts to 94%. Subsequently, the amplified Nb library was cloned into the C-terminally tagged split-YFP1 BiFC vector using the restriction enzymes HindIII and ClaI.

Fluorescence Associated Cell Sorting (FACS) Based on BiFC
To select Nbs recognizing CCR7, HEK293 cells stably expressing CCR7-YFP2 were transiently transfected with pcDNA3-Nb-library-YFP1. Twenty-four hours after transfection, cells were stimulated with 0.5 µg/mL CCL19 (representing the optimal concentration for inducing cell migration [33,34]) for 20 min at 37 • C and 5 % CO 2 . Reconstitution of the two non-fluorescent proteins to native YFP indicating interaction of the Nb with CCR7 was measured by flow cytometry using FACS Aria IIu and the FACSDiva 6 software (BD Biosciences, Franklin Lakes, NJ, USA). BiFC-positive cells were FACS sorted. Beforehand, we conducted a BiFC-negative sorting of HEK293 cells transiently co-expressing pEYFP-N1-β 2 AR-YFP2 and pcDNA3-Nb-library-YFP1, which were stimulated with 10 µM isoproterenol for 20 min at 37 • C and 5 % CO 2 , in order to reduce the number of Nbs recognizing β 2 AR. Flow cytometric data were analyzed using the FlowJo10 software (BD Biosciences).

Isolation of Nbs Interacting with CCR7
Plasmids coding for Nbs interacting with CCR7 in the BiFC system were isolated from FACS sorted, transiently transfected HEK293 cells using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol (Purification of total DNA from Animal Blood or Cells, Spin-Column Protocol). Isolated plasmid DNA was solved in 200 µl ddH 2 O and subsequently transformed into electro-competent E. coli (DH5α) by electroporation. Briefly, approximately 3.5 µg DNA were each added to 500 µl electro-competent E. coli and transferred into a pre-cooled electroporation cuvette (0.4 cm gap) (Bio-Rad, Hercules, CA, USA) on ice. After 2 min, electroporation was carried out using the Gene Pulser Xcell Electroporation System (Bio-Rad) applying 3 kV, 25 µF, 200 Ω for~5 ms. Immediately afterwards, 1 mL of SOC medium was added to the bacteria, transferred into a new 2 mL Eppendorf tube and incubated for 1 h at 37 • C while shaking with 450 rpm. Bacteria (200 µl each) were plated on selective agar (LB medium containing 100 µg/mL ampicillin) plates (Ø 14.5 cm) and incubated overnight at 37 • C. The next day, 96 single colonies were picked from one plate and each inoculated in 5 mL selective LB medium. After incubation overnight at 37 • C and constant shaking with 180 rpm, plasmids coding for single Nb clones were isolated from E. coli using the NucleoSpin ® Plasmid Miniprep Kit (Macherey-Nagel, Düren, Germany), as specified by the manufacturer (protocol for isolation of high copy plasmid DNA from E. coli). Additionally, to generate a new library containing Nbs recognizing CCR7, the remaining colonies were harvested from all plates using a cell scratcher and a sufficient amount of medium (8 mL). The resuspended colonies were added to 1 l (total) selective LB medium. Plasmids coding for various Nb clones were isolated from the E. coli culture without any further amplification using the NucleoBond ® Xtra Midi Kit (Macherey-Nagel) following the manufacturer's instructions (protocol for high copy plasmid purification (Midi)).

Flow Cytometry Analysis of CCR7-Recognizing Nbs
HEK293 cells were transiently transfected either with CCR7-YFP2 or β 2 AR-YFP2 and various single Nb clones (pcDNA3-Nb-YFP1) and analyzed regarding YFP reconstitution by flow cytometry. Twenty-four hours after transfection, cells were stimulated either with 0.5 µg/mL CCL19 or 10 µM isoproterenol for 20 min. Cells were fixed in 4% formaldehyde (Polysciences, Inc., Warrington, PA, USA) for 15 min at RT. After addition of PBG (3% BSA and 20 mM Glycine in PBS), cells were detached, washed twice with PBS, filtered (70 µm cell strainer) (BD Biosciences) and investigated on a LSRII flow cytometer (BD Biosciences) for BiFC. Flow cytometric data were analyzed using the FlowJo10 software (BD Biosciences).

Split-Luciferase Complementation Assay
The split-luciferase complementation assay was performed to investigate direct interaction between β 2 AR-SmBiT/ CCR7-SmBiT and individual Nb-LgBiT clones. Pilot experiments using different plasmid ratios (2:1, 1:1, 1:2, 1:3) revealed best and most reliable results with minimal background at a plasmid ratio of 1:1 (0.5µg each). Twenty-four hours after transient transfection in a 1:1 plasmid ratio, approximately 6*10 5 of transfected HEK293 cells were resuspended in 600 µl PBSG (0.05% glucose in PBS) and for each measurement, 80 µl of the cell suspension were transferred onto a 96 well 1 2 area plate as technical duplicates. After addition of the luminescence substrate coelenterazine H (5 µM), the intensity of luminescence signals was measured on a Spark ® Multimode Microplate Reader (Tecan, Männedorf, Switzerland) for 10 min. Subsequently, 10 µM isoproterenol, 1.5 µg/mL CCL19 or PBS was added to the cells and the recording of luminescence signals was continued for 20 min. The highest luminescence signal detected 7 min after stimulation (cycle 34) for the interaction of β 2 AR-SmBiT with Nb80-LgBiT/ CCR7-SmBiT with Nb1-LgBiT was set to 100% and further compared to the luminescence signals of the other Nb-LgBiT clones.