Nanobodies detecting and modulating GPCRs outside in and inside out

Heukers, Raimond; Groof, Timo W.M. De; Smit, Martine J.

doi:10.1016/j.ceb.2019.01.003

Cited by 50 publications

(49 citation statements)

References 76 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…Subsequently, Nb80 fused to GFP was used as a conformational biosensor to monitor activation of β 2 AR revealing presence of active β 2 AR at the plasma membrane as well as within membranes of early endosomes [22]. As Nbs can be easily produced as recombinant minimal-sized, single domain protein harboring the full antigen binding capacity, in combination with a compact prolate shape and ideal biochemical characteristics, such as solubility, thermal and conformational stability, Nbs represent a promising tool not only to study GPCR structures and functions but also in cancer diagnosis and therapy [23,24,25].…”

Section: Introductionmentioning

confidence: 99%

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Engineering of Nanobodies Recognizing the Human Chemokine Receptor CCR7

Jakobs

Spannagel

Purvanov

et al. 2019

IJMS

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering of Nanobodies Recognizing the Human Chemokine Receptor CCR7

Jakobs

Spannagel

Purvanov

et al. 2019

IJMS

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“…Nanobodies have been used previously to stabilise GPCRs and investigate membrane protein conformations to elucidate new aspects of GPCR function (Zimmermann et al, 2018;Heukers et al, 2018;De Groof et al, 2019a;Heukers et al, 2019). For example, nanobodies recognizing active GPCRs have been used to monitor G protein-mediated signalling after receptor internalization (Irranejad et al, 2013, Stoeber et al, 2018.…”

Section: Discussionmentioning

confidence: 99%

Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Soave

Heukers

Kellam

et al. 2020

Preprint

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max 150 words)Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognises the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C-terminus to the 11-amino acid HiBiT tag (VUN400-HiBiT) which complements to LgBiT protein, forming a full length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect specific conformations of CXCR4. These data demonstrate that the high specificity offered by extracellular-targeted nanobodies can be utilised to probe receptor pharmacology.

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“…As an alternative to the small molecules, more recent research has been focusing on finding GPCR-targeting antibodies and fragments thereof. In particular, nanobodies (antibody fragments derived from the heavy chain-only antibodies of camelids) have gained a lot of interest because of their ability to recognize and stabilize the nonlinear, conformational epitopes typically found of GPCRs (De Groof et al, 2019a;Heukers et al, 2019). These characteristics have been instrumental for the generation of GPCR crystals, thereby allowing the reconstruction of GPCR structures.…”

Section: Viral G Protein-coupled Receptor-targeting Nanobodiesmentioning

confidence: 99%