Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasionin vitroand metastasisin vivo

Kenneth A. Botkjaer,Salvatore Santamaria,Aneesh Karatt-Vellatt,Yoshifumi Itoh,John McCafferty,Henrik J. Ditzel,Mikkel G. Terp,Peter A. Andreasen,Gillian Murphy,Hang Fai Kwok

doi:10.18632/oncotarget.7780

Abstract

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.

Highlights

Membrane-type 1 matrix metalloproteinase (MT1MMP) is a type I transmembrane proteinase thought to be a major effector of pericellular proteolysis in a wide range of cell types [1]
Solution phase selection was carried out using biotinylated pro-MT1-MMP E240A antigen and streptavidin-coated agarose beads, which pulled down antibody fragment clones binding to pro-MT1-MMP
To identify the mechanism underlying the difference in size of the primary tumors and the metastasis in mice treated with MT1-MMP Fc-single chain variable fragment (scFv)’s versus those treated with control Fc-scFv DES, we examined the morphology of primary tumors and metastasis

Summary

Introduction

Membrane-type 1 matrix metalloproteinase (MT1MMP) is a type I transmembrane proteinase thought to be a major effector of pericellular proteolysis in a wide range of cell types [1]. The ability of MT1-MMP to cleave various extracellular matrix (ECM) components, including types I, II and III collagens, fibrin, and laminin 1 and 5, reflect its potential activities during cell invasion of ECM. It can regulate cell signaling functions in response to the changing pericellular environment by the modulation of cell surface receptors, such as the αv and α5 integrins and the ectodomains of syndecan and CD44, as well as some growth factors. It drives the migration and function of autoreactive T-cells [15] and of macrophages in peripheral nerve injury [16], suggesting that this pericellular protease plays significant role in a number of pathological processes and should be considered a viable target for therapeutic interventions

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Results

Conclusion