Utilizing Bioorthogonal Chemistry and Site-Specific PEGylation for Improving the Pharmacokinetic Properties of N-TIMP2

Hezi Hayun haunh@post.bgu.ac.il ^1,2 Niv Papo ^1,2 Eyal Arbely ^2,3,4

¹Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel
²National Institute of Biotechnology, Ben-Gurion University of the Negev, Beer-Sheva, Israel
³Department of Life-Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
⁴Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Matrix metalloproteinases (MMPs) are a family of enzymes that regulate the degradation of the extracellular matrix and its components. These enzymes are regulated by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs), and imbalance in MMP`s activity might lead to a variety of diseases, including cancer.

N-TIMP2_D, a variant of N-TIMP2 that was developed in our lab, showed a great potential as an anti-cancer therapeutic drug, as it has a ~900-fold improved affinity towards MMP-14 compared to the WT and up to 16,000-fold greater specificity towards MMP-14, relatively to other MMPs. However, its short circulation half-life makes a serious limitation for using it as a therapeutic drug. Therefore, we decided to PEGylate N-TIMP2_D in order to increase its size and to improve its pharmacokinetics properties.

The common approaches for protein PEGylation include the conjugation of PEG molecules to the canonical amino acids in a random way that targets all amino acids of the same type, which can be undesired. We have decided to use bioorthogonal chemistry and to incorporate a non-canonical amino acid into N-TIMP2_D, allowing a site-specific PEGylation and a better control for this modification.

We are currently in the process of incorporating the non-canonical amino acid propargyl lysine into N-TIMP2_D in different positions. These residues are aimed to be conjugated with PEG-azide (5K and 20K) in a click chemistry reaction, followed by examination of the inhibition activity towards MMP-14 as well as the pharmacokinetic properties of the modified variants in in vitro and in vivo studies.