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Published on: September 30, 2019
Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display
Jiping Zhang1, Matthildi Valianou, Heidi Simmons
1Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111-2497, USA.
Abstract:
Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.
Insights
Researchers identified the first single-chain variable fragment (scFv) antibody, E3, that inhibits Fibroblast Activation Protein (FAP) activity. This discovery offers a potential new strategy to disrupt FAP
Area of Science:
- Biochemistry
- Immunology
- Oncology
Background:
- Fibroblast Activation Protein (FAP) is a protease found on tumor fibroblasts, crucial for cancer progression, growth, and metastasis.
- FAP's enzymatic activity presents a promising therapeutic target for cancer treatment.
Purpose of the Study:
- To identify and characterize novel inhibitory antibodies targeting FAP.
- To develop potential therapeutic agents against FAP-driven cancer growth and metastasis.
Main Methods:
- Phage display technology was employed to isolate single-chain variable fragments (scFvs) with FAP-binding capabilities.
- Surface plasmon resonance and flow cytometry were used to characterize antibody binding affinity and specificity.
- Functional assays assessed the inhibitory potential of identified scFvs against FAP enzymatic activity.
Main Results:
- Fifty-three anti-FAP scFvs were identified, with one inhibitory antibody, E3, demonstrating 35% attenuation of FAP activity.
- Yeast affinity maturation yielded a mutant E3 scFv with 4-fold higher affinity and 3-fold enhanced FAP inhibition.
- Both E3 and its mutant significantly reduced fibronectin fiber orientation in 3D cell matrix formation.
Conclusions:
- The first FAP-inhibiting scFv antibody (E3) and its high-affinity mutant have been successfully identified and characterized.
- These inhibitory anti-FAP scFvs show potential for disrupting tumor invasion and metastasis by targeting FAP function.
- This work provides a foundation for developing novel antibody-based therapeutics for FAP-expressing cancers.
