PROJECT SUMMARY/ABSTRACT
The MMPs have long been recognized as potential targets for cancer therapy, but drugs developed to target
these enzymes have been unsuccessful. A primary reason has been inadequate selectivity, since most MMP
inhibitors cannot discriminate among MMPs that drive cancer progression and other MMPs that prevent cancer
progression. We have recently developed a new approach, expertise, and methodology for engineering much
more highly selective MMP inhibitors based on a human protein, tissue inhibitor of metalloproteinases-2
(TIMP2). In our recently published work, we have created an engineered variant of the TIMP2 N-terminal
domain (N-TIMP2) with greatly improved selectivity toward MMP-9, an enzyme critically involved in triple-
negative breast cancer (TNBC) progression and metastasis. In preliminary studies, we find that this prototype
inhibitor shows enhanced activity for blocking TNBC cellular invasion. We propose to further engineer N-
TIMP2 for increased selectivity toward MMP-9 and also for enhanced affinity toward a3ß1 integrin, a second
natural target of TIMP2 through which TIMP2 mediates inhibition of tumor growth. We will define the structural
basis for selective MMP binding of engineered N-TIMP2 variants to enable yet greater molecular
improvements, and we will evaluate the therapeutic potential of these engineered proteins in multiple
complementary preclinical models of TNBC. In Aim 1, we will use a combination of structural insights,
computational design and yeast surface display (YSD) technology to engineer N-TIMP2, further optimizing
selectivity toward MMP-9 and enhancing beneficial integrin binding activity. In Aim 2, we will elucidate
structures of the engineered proteins with target and anti-target MMPs using X-ray crystallography, to uncover
the structural basis for engineered selectivity and to facilitate yet greater refinements of our engineering
platform and our selective MMP-9 inhibitors. In Aim 3, we will use complementary mouse orthotopic,
transgenic, and patient-derived xenograft (PDX) models of TNBC to evaluate the utility of engineered N-TIMP2
variants as a therapeutic strategy in TNBC, and identify candidate biomarkers of response with potential for
directing this therapeutic approach to patients who will most benefit from it. Our proposal is both conceptually
and technically innovative in the combination of approaches toward generating novel protein therapeutics. The
proposed research is highly significant because it has substantial potential to develop an entirely new
approach for targeted treatment of TNBC by selectively inhibiting MMP-9, a well-validated target with key roles
in tumor growth, invasion, metastasis, and angiogenesis.