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Tuning the Structural Properties of a Single-Domain Antibody Scaffold for Improved Fibroblast Activation Protein
Joseph P Gallant1,2, Kendahl L Ott1,2, Ohyun Kwon3
1Molecular and Cellular Pharmacology Program, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin53705, United States.
Molecular Pharmaceutics
|July 21, 2026
Summary
Camelid single-domain antibodies targeting fibroblast activation protein (FAP) show promise for cancer theranostics. Engineering these antibodies into different formats (monomer, dimer, Fc-fusion) optimized tumor targeting and retention for improved imaging and potential therapy.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Fibroblast activation protein (FAP) is selectively expressed on cancer-associated fibroblasts (CAFs), making it a key target for cancer theranostics.
- While small-molecule FAP inhibitors (FAPIs) exist, biologics for FAP targeting are less explored.
- Camelid-derived single-domain antibodies (VHHs) offer high affinity and engineering flexibility for targeted delivery.
Purpose of the Study:
- To develop and characterize novel anti-FAP VHH-based constructs for cancer theranostics.
- To investigate the impact of valency and molecular weight on FAP-targeting VHHs' in vivo properties.
- To evaluate the potential of engineered VHH formats for positron emission tomography (PET) imaging of FAP-positive tumors.
Main Methods:
- Identification of a novel anti-FAP VHH (F7) using phage display.
- Engineering F7 into monomer (F7), tethered dimer (F7D), and Fc-fusion (F7-Fc) formats.
- In vivo evaluation using FAP-positive xenograft models and PET imaging, including pharmacokinetic analysis and absorbed dose calculations.
Main Results:
- All three F7 formats demonstrated FAP specificity, with bivalent constructs achieving picomolar affinity.
- PET imaging revealed distinct pharmacokinetic profiles: F7 showed rapid uptake/clearance, F7D offered high tumor-to-blood ratios, and F7-Fc provided the highest tumor uptake for longitudinal imaging.
- Absorbed dose calculations indicated significantly higher radiation doses to tumors compared to normal tissues.
Conclusions:
- Engineering VHH scaffold size and valency can significantly improve biodistribution and tumor retention for FAP-targeting agents.
- F7-based constructs, particularly F7D and F7-Fc, represent promising targeting vectors for FAP-positive cancer theranostics.
- This study highlights the potential of VHH engineering for optimizing targeted cancer therapies and diagnostics.
