Utility of Glycosylated TIMP3 molecules: Inhibition of MMPs and TACE to improve cardiac function in rat myocardial infarct model

Abstract

Tissue Inhibitor of Metalloproteinase 3 (TIMP3) is a secreted protein that has a great utility to inhibit elevated metalloproteinase (MMP) activity in injured tissues including infarcted cardiac tissue, inflamed vessels, and joint cartilages. An imbalance between TIMP3 and active MMP levels in the local tissue area may cause worsening of disease progression. To counter balance elevated MMP levels, exogenous administration of TIMP3 appeared to be beneficial in preclinical studies. However, the current form of WT‐TIMP3 molecule has a limitation to be a therapeutic candidate due to low production yield, short plasma half‐life, injection site retention, and difficulty in delivery, etc. We have engineered TIMP3 molecules by adding extra glycosylation sites or fusing with albumin, Fc, and antibody to improve pharmacokinetic properties. In general, the C‐terminal fusion of TIMP3 improved expression and production in mammalian cells and extended half‐lives dramatically 5‐20 folds. Of note, a site‐specific glycosylation at K22S/F34N resulted in a higher level of expression and better cardiac function compared to other fusion proteins in the context of left ventricle ejection fraction (LVEF) changes in a rat myocardial infarction model. It appeared that cardiac efficacy depends on a high ECM binding affinity, in which K22S/F34N and N‐TIMP3 showed a higher binding to the ECM compared to other engineered molecules. In conclusion, we found that the ECM binding and sustained residence of injected TIMP3 molecules are important for cardiac tissue localization and inhibition of adverse remodeling activity.

Biology of Tissue Inhibitor of Metalloproteinase 3 (TIMP3), and Its Therapeutic Implications in Cardiovascular Pathology

“…TIMP3v2 or TIMP3v82 were injected into the infarcted myocardium after 3 h of LAD ligation in rats. Both could improve cardiac function at 3 days post-MI, but only TIMP3v82 had lasting effects until 7 days post-MI (Chintalgattu et al, 2018).…”

“…Further, a marked but transient rise in MMP activity in the early post-MI phase was found to be responsible for the adverse remodeling and increased rate of LV rupture in Timp3 −/− mice, since early treatment with a broad-spectrum MMP inhibitor ameliorated the adverse LV remodeling and rupture (Kandalam et al, 2010). On the other hand, targeted overexpression of TIMP3 or delivery of recombinant TIMP3 protein into the myocardium also improved the infarcted myocardium and heart function through suppression of the MMP activity and inflammation, and promoting angiogenesis (Eckhouse et al, 2014;Purcell et al, 2014Purcell et al, , 2018Barlow et al, 2017;Takawale et al, 2017;Chintalgattu et al, 2018;Thorn et al, 2019).…”

“…Besides the recombinant wild-type TIMP3 protein, utility of engineered TIMP3 molecule has also been explored. These modifications include addition of extra glycosylation sites, PEGylation (polyethylene glycol conjugation), and fusion with Fc, human serum albumin (HSA), antibody (Ab), or latency-associated peptide (LAP) (Chintalgattu et al, 2018;Alberts et al, 2019). To sequester TIMP3 in the extracellular space, TIMP3 could also be mutated (K26A/K45A and K42A/K110A) to disrupt its interaction with LRP-1 and the subsequent endocytosis (Doherty et al, 2016).…”

“…To sequester TIMP3 in the extracellular space, TIMP3 could also be mutated (K26A/K45A and K42A/K110A) to disrupt its interaction with LRP-1 and the subsequent endocytosis (Doherty et al, 2016). Two engineered mutant TIMP3 molecules with extra glycosylation sites, TIMP3v2 (K22S/F34N) and TIMP3v82 (H55N/Q57T/K71N/E73T/D87N/K89T/R115T), have a higher expression yield in a Chinese hamster ovary cell line and longer rat serum half-life than the wild-type TIMP3 (Chintalgattu et al, 2018). TIMP3v2 or TIMP3v82 were injected into the infarcted myocardium after 3 h of LAD ligation in rats.…”

Disturbed balance in the expression of MMP9 and TIMP3 in cerebral amyloid angiopathy-related intracerebral haemorrhage

“…Tissue inhibitors of metalloproteinases (TIMPs) regulate the activity of MMPs and may therefore be indirectly involved in the regulation of ECM degradation and thus the development of haemorrhages. TIMP3 can form a stable complex with pro-MMP9 [29], and has been shown to inhibit MMP9 activity [8]. Using immunohistochemistry (IHC), next to other techniques, increased cerebrovascular TIMP3 accumulation in brain vessels has been detected in patients with CAA [24], and patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) [27], a hereditary form of cerebral small vessel disease.…”