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Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)
Published on: September 17, 2019
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.
Abstract:
Fibroblast activation protein (FAP) is an attractive target for the development of cancer theranostics due to its selective expression on cancer-associated fibroblasts (CAFs). While a number of small-molecule FAP inhibitors (FAPIs) have been developed, few biologics have been investigated as FAP-targeting vectors. Camelid-derived single-domain antibodies, or variable-heavy-heavy domains (VHHs), offer a compelling alternative, combining high affinity with versatile engineering options. In this study, we first identified a novel anti-FAP VHH, F7, from an affinity-matured camelid phage display library. To investigate how valency and molecular weight affected target engagement and in vivo properties, F7 was engineered into three formats: a monomer (F7), a tethered dimer (F7D), and an Fc-fusion protein (F7-Fc). All three were specific for FAP with the two bivalent constructs demonstrating picomolar affinity. Positron emission tomography imaging in FAP-positive xenograft models revealed distinct pharmacokinetic profiles across constructs, with notable differences in tumor uptake and clearance. F7 had rapid uptake and clearance, resulting in significantly higher tumor uptake than FAPI-46. Low molecular weight bivalent F7D demonstrated similar kinetics but was retained by the tumor, resulting in a high tumor-to-blood ratio with secondary uptake limited to clearance organs. The largest construct, F7-Fc, resulted in the highest tumor uptake and allowed for longitudinal imaging. Absorbed dose calculations confirmed that tumors received significantly higher radiation doses compared to normal tissues. These findings demonstrate that tuning VHH scaffold size and valency can improve biodistribution and retention, establishing F7-based constructs as promising targeting vectors for FAP.
Insights
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.
