Identification and characterization of Nanobodies targeting the EphA4 receptor.

The Journal of Biological Chemistry
Lies SchoonaertW Robberecht

Abstract

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. We identified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb 39 and Nb 53 inhibited ephrin-induced phosphorylation of the EphA4 protein in a cell-based assay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3...Continue Reading

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Citations

Apr 10, 2020·Applied Microbiology and Biotechnology·Zhixin FangJianxiang Qiu
Aug 15, 2020·Frontiers in Oncology·Emily Y Yang, Khalid Shah
Apr 30, 2020·Expert Opinion on Investigational Drugs·Oscar J BuckensGodefridus J Peters
Jul 8, 2020·Pharmaceuticals·Sarah BaudetXavier Nicol
Nov 6, 2020·Current Protocols in Neuroscience·James S Trimmer
Jan 17, 2021·Molecular Immunology·Wenyi WangChangan Jiang

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Methods Mentioned

BETA
ELISA
immunoprecipitation
surface plasmon resonance
chip
AlphaScreen
antisense oligonucleotides
isothermal titration calorimetry
PCR
AlphaLISA

Software Mentioned

Zeiss AxioVision V
Biacore T200
AlphaScreen

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