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Cryo-electron microscopy reveals a single domain antibody with a unique binding epitope on fibroblast activation protein alpha
Preprint   Open access

Cryo-electron microscopy reveals a single domain antibody with a unique binding epitope on fibroblast activation protein alpha

Zhen Xu, Akesh Sinha, Darpan N Pandya, Nicholas J Schnicker and Thaddeus J Wadas
bioRxiv
Cold Spring Harbor Laboratory
10/19/2024
DOI: 10.1101/2024.10.18.619146
PMCID: PMC11507940
PMID: 39463996
url
https://doi.org/10.1101/2024.10.18.619146View
Preprint (Author's original)This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this unique expression profile, designing effective diagnostic and therapeutic agents that effectively target this biomarker remain elusive. Here we report the structural characterization of the interaction between a novel single domain antibody (sdAbs), I3, and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 Å and contained two distinct populations; one I3 bound and two I3 molecules bound to the FAP dimer. In both cases, the sdAbs bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this report describes the rational mutation of specific residues within the complementarity determining region 3 (CDR3) loop to enhance affinity and selectivity of the I3 molecule for FAP. This report represents the first sdAb-FAP structure to be described in the literature.Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this unique expression profile, designing effective diagnostic and therapeutic agents that effectively target this biomarker remain elusive. Here we report the structural characterization of the interaction between a novel single domain antibody (sdAbs), I3, and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 Å and contained two distinct populations; one I3 bound and two I3 molecules bound to the FAP dimer. In both cases, the sdAbs bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this report describes the rational mutation of specific residues within the complementarity determining region 3 (CDR3) loop to enhance affinity and selectivity of the I3 molecule for FAP. This report represents the first sdAb-FAP structure to be described in the literature.

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