Engineering of Nanobodies Recognizing the Human Chemokine Receptor CCR7

Autor: Barbara D. Jakobs, Lisa Spannagel, Vladimir Purvanov, Edith Uetz-von Allmen, Christoph Matti, Daniel F. Legler
Rok vydání: 2019
Předmět:
Zdroj: International Journal of Molecular Sciences
Volume 20
Issue 10
International Journal of Molecular Sciences, Vol 20, Iss 10, p 2597 (2019)
ISSN: 1422-0067
Popis: 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 &beta
2-adrenergic receptor (&beta
2AR) and by specifically randomizing two segments within complementarity determining region 1 (CDR1) and CDR3 of Nb80 known to interact with &beta
2AR. 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.
Databáze: OpenAIRE
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