Abstract
Targeting dysregulated protease expression and/or abnormal substrate proteolysis, highly selective inhibition of pathogenic proteases by monoclonal antibodies (mAbs) presents an attractive therapeutic approach for the treatment of diseases including cancer. Herein, we report a functional selection method for protease inhibitory mAbs by periplasmic co-expression of three recombinant proteins—a protease of interest, an antibody Fab library, and a modified β-lactamase TEM-1. We validate this approach by isolation of highly selective and potent mAbs inhibiting human matrix metalloproteinase 9 (MMP9).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Turk B, Turk D, Turk V (2012) Protease signalling: the cutting edge. EMBO J 31:1630–1643
Deu E, Verdoes M, Bogyo M (2012) New approaches for dissecting protease functions to improve probe development and drug discovery. Nat Struct Mol Biol 19:9–16
López-Otín C, Bond JS (2008) Proteases: multifunctional enzymes in life and disease. J Biol Chem 283:30433–30437
Docherty AJ, Crabbe T, O’Connell JP et al (2003) Proteases as drug targets. Biochem Soc Symp 2003:147–161
Turk B (2006) Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 5:785–799
Drag M, Salvesen GS (2010) Emerging principles in protease-based drug discovery. Nat Rev Drug Discov 9:690–701
Ganesan R, Eigenbrot C, Kirchhofer D (2010) Structural and mechanistic insight into how antibodies inhibit serine proteases. Biochem J 430:179–189
Chavarria-Smith J, Chiu CPC, Jackman JK et al (2022) Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis. Sci Transl Med 14:eabp9159
Nam DH, Rodriguez C, Remacle AG et al (2016) Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries. Proc Natl Acad Sci U S A 113:14970–14975
Nam DH, Ge X (2018) Generation of highly selective MMP antibody inhibitors. Methods Mol Biol (Clifton, NJ) 1731:307–324
Lopez T, Mustafa Z, Chen C et al (2019) Functional selection of protease inhibitory antibodies. Proc Natl Acad Sci U S A 116:16314–16319
Hayhurst A, Happe S, Mabry R et al (2003) Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis. J Immunol Methods 276:185–196
Nam DH, Lee KB, Ge X (2018) Functional production of catalytic domains of human MMPs in Escherichia coli periplasm. Methods Mol Biol (Clifton, NJ) 1731:65–72
Lee KB, Nam DH, Nuhn JAM et al (2017) Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli. Microb Cell Factories 16:73
Galarneau A, Primeau M, Trudeau LE et al (2002) Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat Biotechnol 20:619–622
Lee KB, Dunn ZS, Lopez T et al (2020) Generation of highly selective monoclonal antibodies inhibiting a recalcitrant protease using decoy designs. Biotechnol Bioeng 117:3664–3676
Nam DH, Lee KB, Kruchowy E et al (2020) Protease inhibition mechanism of camelid-like synthetic human antibodies. Biochemistry 59:3802–3812
Lee KB, Dunn Z, Ge X (2019) Reducing proteolytic liability of a MMP-14 inhibitory antibody by site-saturation mutagenesis. Protein Sci 28:643–653
Acknowledgments
This work was supported by Cancer Therapeutics Training Program Fellowship to K.B.L. (CPRIT RP210043) and NIH R35GM141089 to X.G.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Lee, K.B., Ge, X. (2024). Generation of Protease Inhibitory Antibodies by Functional In Vivo Selection. In: Santamaria, S. (eds) Proteases and Cancer. Methods in Molecular Biology, vol 2747. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3589-6_19
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3589-6_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3588-9
Online ISBN: 978-1-0716-3589-6
eBook Packages: Springer Protocols