A monoclonal antibody interferes with TIMP-2 binding and incapacitates the MMP-2-activating function of multifunctional, pro-tumorigenic MMP-14/MT1–MMP

Abstract

Matrix metalloproteinases (MMPs) and, especially membrane type 1 (MT1)-MMP/MMP-14, are promising drug targets in malignancies. In contrast with multiple small-molecule and protein pan-inhibitors of MT1–MMP cleavage activity, the murine 9E8 monoclonal antibody targets the MMP-2-activating function of cellular MT1–MMP alone, rather than the general proteolytic activity and the pro-migratory function of MT1–MMP. Furthermore, the antibody does not interact in any detectable manner with other members of the membrane type (MT)-MMP family. The mechanism of this selectivity remained unknown. Using mutagenesis, binding and activity assays, and modeling in silico, we have demonstrated that the 9E8 antibody recognizes the MT-loop structure, an eight residue insertion that is specific for MT–MMPs and that is distant from the MT1–MMP active site. The binding of the 9E8 antibody to the MT-loop, however, prevents tissue inhibitor of metalloproteinases-2 (TIMP-2) association with MT1–MMP. As a result, the 9E8 antibody incapacitates the TIMP-2-dependent MMP-2-activating function alone rather than the general enzymatic activity of human MT1–MMP. The specific function of the 9E8 antibody we determined directly supports an essential, albeit paradoxical, role of the protein inhibitor (TIMP-2) in MMP-2 activation via a unique membrane-tethered mechanism. In this mechanism, the formation of a tri-molecular MT1–MMPTIMP-2MMP-2 complex is required for both the capture of the soluble MMP-2 proenzyme by cells and then its well-controlled conversion into the mature MMP-2 enzyme. In sum, understanding of the structural requirements for the 9E8 antibody specificity may pave the way for the focused design of the inhibitory antibodies against other individual MMPs.

Ginkgo biloba extract inhibits oxidized low-density lipoprotein (oxLDL)-induced matrix metalloproteinase activation by the modulation of the lectin-like oxLDL receptor 1-regulated signaling pathway in human umbilical vein endothelial cells

“…TIMP-1 is the inhibitor of MMP. 16 We confirmed pretreatment of GbE reverses oxLDL-inhibited TIMP-1 expression in messenger RNA (Fig 1, B) and protein (Fig 1, C and E). We also used ELISA and MMPs activity assay to test the MMP-1, MMP-2, and MMP-3 concentration and activity in medium (Fig 1, F and G).…”

“…Human plasma LDL was obtained from Sigma-Aldrich. Copper-modified LDL (1 mg protein/mL) was prepared by exposing LDL to 10 mM CuSO 4 for 16 hours at 37 C. 16 After oxidation, the amount of thiobarbituric acid reactive substances in LDL ranged from 15 to 20 nmol/mg LDL.…”

Overview of MMP Biology and Gene Associations in Human Diseases

“…Up to date, functional blocking antibodies that specifically target MT-MMPs have been developed. What is the most important, it seems that antibodies could target specific function of MMP rather than its broad proteolytic activity [90].…”

New approaches to selectively target cancer-associated matrix metalloproteinase activity

“…Therefore, partial inhibitors of MMP-14 that prevent cancer progression but do not impact normal processes over the long term may be more clinically useful. In this regard, the monoclonal antibody, 9E8, blocks MMP-14 activation of MMP-2 but spares MMP-14 promigratory effects and proteolytic functions because the epitope is an 8-residue loop that is distal from the catalytic domain [77]. The catalytic domain of other nonmembrane bound MMPs can also be targeted using monoclonal antibodies (Table 3).…”