Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo
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Kenneth A. Botkjaer1, Hang Fai Kwok1,8, Mikkel G. Terp2, Aneesh Karatt-Vellatt3, Salvatore Santamaria4, John McCafferty3, Peter A. Andreasen5,6, Yoshifumi Itoh4, Henrik J. Ditzel2,7, Gillian Murphy1
1Department of Oncology, University of Cambridge, Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Cambridge CB2 0RE, U.K
2Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark
3IONTAS Ltd., Babraham Research Campus, Cambridge CB22 3AT, U.K
4Kennedy Institute of Rheumatology, University of Oxford, Headington, Oxford OX3 7FY, U.K
5Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
6Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Aarhus, Denmark
7Department of Oncology, Odense University Hospital, Odense, Denmark
8Faculty of Health Sciences, University of Macau, Taipa, Macau SAR
Kenneth A. Botkjaer, e-mail: Kenneth.firstname.lastname@example.org
Hang Fai Kwok, e-mail: email@example.com
Gillian Murphy, e-mail: firstname.lastname@example.org
Keywords: MT1-MMP, antibody, non-catalytic sites, in vivo targeting, metastasis
Received: September 21, 2015 Accepted: January 23, 2016 Published: February 27, 2016
The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332-fold with the ability to interfere with cell-surface MT1-MMP functions, displaying IC50 values down to 5 nM. Importantly, the new inhibitors were able to inhibit collagen invasion by tumor-cells in vitro and in vivo primary tumor growth and metastasis of MDA-MB-231 cells in a mouse orthotopic xenograft model. Herein is the first demonstration that an inhibitory antibody targeting sites outside the catalytic cleft of MT1-MMP can effectively abrogate its in vivo activity during tumorigenesis and metastasis.
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