Targeting the MMP-9/CD44 axis for cancer treatment

Matrix-metalloproteinase-9 (MMP-9) and CD44 are two known proteins that are highly involved in tumor progression. These proteins have many pro-tumor cross-talks between them, such as cleavage of CD44 by MMP-9 that stimulates cells motility, and MMP-9 localization on CD44 via its hemopexin domain (PEX), which increases cell invasion. Moreover, CD44 regulates MMP-9 expression.

MMP-9 is a member of the MMP family, an ECM degrading enzymes. The ECM degradation leads to growth factor release and activation, invasion and metastasis. MMPs are regulated by their natural broad spectrum inhibitor, tissue inhibitors of matrix metalloproteinase-2 (TIMP)-2. Since MMP-9 is involved in tumor progression and other pathological conditions, such as osteoporosis and heart failure, there is a necessity for an effective and more specific inhibitor for this enzyme.

In order to generate high affinity inhibitor towards MMP-9, we utilized the N-terminal domain of TIMP2 (N-TIMP2) as the starting point for a yeast surface display system (YSD) library. This library was screened for MMP-9 high binders, which led to the finding of C9 variants that contains 7 mutations. This variant exhibited stronger inhibition ability of MMP-9 compared to the N-TIMP2_WT.

In order to increase the specificity and the inhibition activity towards MMP-9, we engineered a bispecific chimera, termed C9-PEX, that can simultaneously target both MMP-9 and its co-localized receptor- CD44. This chimera composed of C9 conjugated to MMP-9 PEX domain via flexible linker. This chimera could compete with MMP-9 localization on the cell surface, inhibit MMP-9 catalytic activity, decrease MMP-9 expression and decrease MAPK phosphorylation which would result in invasion inhibition of cancerous cells.