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

Zhen Xu ¹, Akesh Sinha ², Darpan N Pandya ²

⁰Protein and Crystallography Facility, University of Iowa, Iowa City, Iowa, 52242, USA.

Biorxiv : the Preprint Server for Biology +

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October 28, 2024

View abstract on PubMed

Summary

Researchers characterized a novel single domain antibody (sdAb) interaction with fibroblast activation protein alpha (FAP). This structural insight provides a foundation for developing new diagnostic and therapeutic FAP-targeting agents.

Area Of Science

Biochemistry
Structural Biology
Immunology

Background

Fibroblast activation protein alpha (FAP) is a protease overexpressed in various pathologies, notably cancer.
Targeting FAP presents opportunities for diagnostics and therapeutics, but effective agents remain elusive.
Understanding FAP interactions is crucial for developing novel biomedical tools.

Purpose Of The Study

To structurally characterize the interaction between a novel single domain antibody (sdAb), I3, and FAP.
To provide a detailed structural basis for FAP-sdAb interactions.
To guide the rational design of improved FAP-targeting agents.

Main Methods

Cryo-electron microscopy (cryo-EM) was employed to determine the structure of the I3-FAP complex.
High-resolution (2.7 Å) reconstructions revealed distinct binding modes of the sdAb to the FAP dimer.
Site-directed mutagenesis of the sdAb's complementarity determining region 3 (CDR3) was performed.

Main Results

The study elucidated the first reported structure of an sdAb bound to FAP.
Two distinct populations were observed: one with one I3 molecule bound and another with two I3 molecules bound to the FAP dimer.
The sdAbs bind to a unique epitope, separate from the FAP active site.
Rational mutations in the CDR3 loop demonstrated potential for enhancing affinity and selectivity.

Conclusions

This work presents the first structural description of an sdAb-FAP complex.
The identified binding site and potential for affinity enhancement offer a promising avenue for FAP-targeted diagnostics and therapeutics.
These findings lay the groundwork for developing next-generation FAP-targeting agents.