Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens

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. Usin...

Full description

Saved in:
Bibliographic Details
Published in:The FASEB journal 2013-02, Vol.27 (2), p.581-589
Main Authors: Zhang, Jiping, Valianou, Matthildi, Simmons, Heidi, Robinson, Matthew K., Lee, Hyung‐Ok, Mullins, Stefanie R., Marasco, Wayne A., Adams, Gregory P., Weiner, Louis M., Cheng, Jonathan D.
Format: Article
Language:English
Subjects:
Animals
Antibody Affinity
Flow Cytometry
Gelatinases - antagonists & inhibitors
Gelatinases - genetics
Gelatinases - immunology
Gelatinases - metabolism
Humans
Membrane Proteins - antagonists & inhibitors
Membrane Proteins - genetics
Membrane Proteins - immunology
Membrane Proteins - metabolism
Mice
Molecular Targeted Therapy
Mutagenesis, Site-Directed
Mutant Proteins - genetics
Mutant Proteins - immunology
Neoplasms - drug therapy
Neoplasms - enzymology
Peptide Library
Recombinant Proteins - antagonists & inhibitors
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Recombinant Proteins - metabolism
Research Communications
Serine Endopeptidases - genetics
Serine Endopeptidases - immunology
Serine Endopeptidases - metabolism
serine protease
Single-Chain Antibodies - genetics
Single-Chain Antibodies - immunology
Single-Chain Antibodies - metabolism
single‐chain variable fragment
Surface Plasmon Resonance
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.—Zhang, J., Valianou, M., Simmons, H., Robinson, M. K., Lee, H.‐O., Mullins, S. R., Marasco, W. A., Adams, G. P., Weiner, L. M., Cheng, J. D. Identification of inhibitory ScFv antibodies targeting fibroblast activation protein utilizing phage display functional screens. FASEB J. 27, 581–589 (2013). www.fasebj.org
ISSN:0892-6638
1530-6860
DOI:10.1096/fj.12-210377