Rational and Combinatorial Approaches to Develop the N-Terminal Domain of TIMP2 with High Specificity towards MMP14 and MMP9 for Cancer Therapy

Matrix Metalloproteases (MMPs), especially MMP14 and MMP9 play a major role in cancer progression, angiogenesis and metastasis due to their ability to degrade the extra cellular matrix components. Though many attempts were made to develop inhibitors for MMPs, lack of specificity of these inhibitors is a major obstacle in developing them as effective anticancer agents. The endogenous tissue inhibitor of matrix metalloproteases 2 (TIMP2) inhibits the MMPs catalytic activity by interacting with the catalytic site of MMPs via its N-terminus. We employed a state of the art directed evolution technique, namely yeast surface display system (YSD), in order to engineer N-TIMP2 variants with high affinity and specificity towards MMP14 and MMP9, respectively. We identified an N-TIMP2 mutant that has high affinity towards MMP14. In in-vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. In order to increase the variant`s half-life in circulation for in-vivo studies the variant is modified by chemical methods of polyethileneglycol addition. In a related project, the N-TIMP2 selective variants are being developed via screening of the focused library for binding to MMP14 and MMP9 applied simultaneously. Selective clone towards MMP14 showed up to 200-fold improved specificity towards MMP14 over MMP9. Clones specifically targeting MMP9 are being produced and will be further evaluated in-vitro and in cell models. Our approach enables us to identify specific N-TIMP2 based inhibitors that can be further utilized for used for cancer stage diagnostics, prognosis assessment and for personalized medicine. Furthermore, the approach can be extended to other MMPs increasing the variability (and availability) of cancer therapeutics.