Development of an inhibitory monoclonal antibody targeting the novel coronary artery disease risk factor ADAMTS-7

Background: Genome-wide association studies (GWAS) identified the ADAMTS7 locus which encodes the extracellular matrix (ECM) protease ADAMTS-7 as a risk factor for coronary artery disease (CAD). Experimental studies in animal models revealed that ADAMTS-7 promotes plaque formation and influences plaque stability via modulation of the ECM. Our group recently identified the endogenous matrix metalloproteinase (MMP) inhibitor TIMP-1 (tissue inhibitor of metalloprotease-1) and its bona fide target MMP-9 as possible contributors to these observations. Inhibiting ADAMTS-7 might therefore be a promising approach in the prevention of CAD. Selective inhibitors are, however, thus far not available.
 
Methods and Results: A construct containing a truncated human ADAMTS-7 incorporating the C-terminal thrombospondin (TSP) 5-8 domains (ADAMTS7_TSP) was used as antigen. The construct was transformed into Agrobacterium tumefaciens, which was then infiltrated into Nicotiana benthamiana leaves to induce recombinant protein production. Purified proteins were then used to stimulate antibody production in mice. Hhybridoma cells that continuously produce monoclonal antibodies (mAb) against ADAMTS-7 were created. We tested generated antibodies for their binding ability to ADAMTS7_TSP using surface plasmon resonance and identified significant binding responses with mAb32.5, yielding a Kd value of 68 nM. Binding of the antibody to murine and human ADAMTS-7 was validated using immunoblotting. To assess the inhibitory properties of monoclonal antibody mAb32 on the interaction between ADAMTS-7 and TIMP-1 time-resolved, we used an assay based on Förster resonance energy transfer (FRET). Protein extracts from transiently transfected HEK293 cells with ADAMTS-7_FLAG and TIMP-1_HA were incubated with donor- (FLAG-cryptate) and acceptor- (HA-d2) fluorophores to induce a baseline FRET signal which serves as readout for the interaction. An antibody specific to an unrelated plant protein that was produced in the similar way, was used as control. We found that mAb32.5 significantly inhibited the ADAMTS-7/TIMP-1 interaction as compared to control (FRET ratios: 0.70±0.14 vs. 1.12±0.29 [a.u.], mAb32.5 vs. control, mean ± s.e.m., n=9, p=5·10^-4) at the concentration of 0.05 g/l. Antibody titration experiments revealed a dose-dependent effect. Next, we sought to investigate the effect of mAb32.5 on Adamts-7 inhibition in modulating plaque progression in vivo using atherosclerotic mice. To that end, Apoe-/- mice were subjected to a Western diet for 4 weeks to initiate plaque formation, followed by a 6-week period where the mice additionally received weekly injections of either mAb32.5 or control (10 mg/kg body weight). Afterwards, the whole aorta was isolated and subjected to en face staining using Oil Red O to quantify atherosclerotic plaques. The aortas from the mAb32-treated group displayed a significant reduction in plaque area as compared to the control group (12.26±2.92 vs. 8.86±2.31 [%] , mAb32.5 vs. control, mean±s.e.m., n=12 each, p=2.7·10^-3).

Conclusion: ADAMTS-7 represents a promising novel CAD target. The monoclonal antibody mAb32.5 recognizes ADAMTS-7 and inhibits the interaction between ADAMTS-7 and TIMP-1. In vivo, treatment with mAb32.5 resulted in decreased plaque burden in an atherosclerotic mouse model. Future experiments will focus on the humanization of mAb32.5 and investigation of its safety and efficacy in large-animal models.