June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Structure-guided GPCR drug development of human single domain camelid antibodies (Nanobody) for autosomal dominant retinitis pigmentosa
Author Affiliations & Notes
  • Arum Wu
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • David Salom
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • John Hong
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • Els Pardon
    VIB-VUB Center for Structural Biology, Vrije Universiteit Brussel, Brussel, Belgium
  • Jan Steyaert
    VIB-VUB Center for Structural Biology, Vrije Universiteit Brussel, Brussel, Belgium
  • Philip David Kiser
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • Krzysztof Palczewski
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Arum Wu None; David Salom None; John Hong None; Els Pardon None; Jan Steyaert None; Philip Kiser None; Krzysztof Palczewski None
  • Footnotes
    Support  VA Grant I01BX004939, NSF Grant CHE-2107713
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 765. doi:
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      Arum Wu, David Salom, John Hong, Els Pardon, Jan Steyaert, Philip David Kiser, Krzysztof Palczewski; Structure-guided GPCR drug development of human single domain camelid antibodies (Nanobody) for autosomal dominant retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2023;64(8):765.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Treatment for autosomal dominant retinitis pigmentosa (adRP) by gene therapy is the most promising option but challenging because adRP is clinically and genetically heterogeneous. There is growing interest to invent therapeutic biologics, including monoclonal antibodies due to the favorable safety profiles and longer half-lives compared to small molecules. Thus, we have developed a series of nanobodies targeting the extracellular surface of bovine rhodopsin, exhibiting therapeutic properties in an adRP cell model. We hypothesized that the mechanism-of-action of the extracellularly binding nanobodies is through the stabilization of the orthosteric site, resulting in the suppression of hydrogen bonding network changes, subsequent allosteric transition, and reduction in the hydrolysis of the agonist from the binding pocket.

Methods : Key epitope residues for nanobody recognition was validated by mutagenic analyses. The effect of an allosterically binding nanobody on Schiff base hydrolysis of agonist was analyzed by liquid chromatographic mass spectrometry and intrinsic tryptophan fluorescence quenching. The biochemical properties of nanobodies in complex with rhodopsin from human retina was analyzed by co-immunoprecipitation and UV-Vis spectroscopy upon photoactivation.

Results : Mutagenic analysis revealed the antigenic nature of extracellular loop 2 and N-terminal glycans, critical for nanobody recognition. Binding of nanobody to the extracellular surface suppressed proton transfer events at the agonist binding site and prevented outward movement of helices five and six – a universal activation event for GPCRs. In a P23H adRP cell model, the hydrolysis of agonist from the mutant opsins was significantly slowed by nanobody treatment. Finally, extracellularly binding human nanobodies also stabilized inactive, photoactivated conformations.

Conclusions : Our extracellularly binding nanobodies identified the role of extracellular loop 2 and N-terminal glycans during the early allosteric transition to inactive, intermediate conformation of a GPCR. The dual action of nanobodies as an allosteric modulator and molecular chaperone is novel for GPCR antibodies, which typically act competitively against ligand binding. These findings suggest a new approach to GPCR antibody drug to modulate activity and become increasingly appealing to the pharmaceutical industry.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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