(471a) Invited Talk: Developing Multi-Inhibitory Protein Therapeutics Targeting ECM Proteins Using Computational and Experimental Protein Engineering and Design Approaches

Our research group is interested in engineering and design of protein scaffolds as potential therapeutics for selective targeting of Metalloproteinases (MPs). MPs including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs) play a multifaceted role in several diseases, such as cancer, neurological disorders, and cardiovascular diseases, and pre-term labor based on their critical role in remodeling the extracellular matrix (ECM) and other proteolytic activities in ECM. Protein-based MP inhibitors offer higher stability and selectivity which is critical for developing efficient therapeutics with low off-target effects. Tissue inhibitors of metalloproteinase (TIMPs), natural inhibitors of matrix metalloproteinases (MMPs), and monoclonal antibodies (mAbs), provide excellent protein scaffolds for engineering selective or multi-specific MP inhibitors. Our research group has engineered and designed MP inhibitors based on TIMPs or single chain fragment (scFv) antibodies by combination of directed evolution, computational modeling, and machine learning approaches.

We use a combination of computational and experimental approaches to design highly selective MP binders and inhibitors. Our group recruits a combination of directed evolution, rational design, computational modeling, and machine learning to unravel the key motifs and residues that drive selective inhibition of MPs, a critical effort to develop new generation of highly selective protein drugs with low off-target effects. The advances in protein engineering techniques as well as computational studies to predict structure and function of proteins provides robust tools to engineer and design MP inhibitors as potential protein therapeutics to treat MP-related diseases. Our group is invested in two main classes of protein inhibitors: TIMPs and antibodies. Here, we report our most recent progress in generating hybrid inhibitors that target specific ECM proteins such as MMP-3, -9, ADAM-17, and VEGFR2, and synthetic antibodies targeting MMP-9 and ADAM-17.

TIMPs have a high level of sequence and structure homology, with a broad range of inhibition selectivity and binding affinity to the family of MMPs. We previously used DNA shuffling between the human TIMP family to generate a minimal TIMP hybrid library to identify the dominant minimal MMP inhibitory regions with higher flexibility and higher tissue penetration features. More recently, we engineered hybrid TIMPs using domain and loop swapping techniques for improving stability and binding selectivity to specific MPs such as MMP-3/-9, ADAM-17, and growth factors such as VEGFR2. For instance, we showed that the insertion of certain loops such as C-connector disrupts expression and binding, while MTL loop insertion improves and/or recovers this effect.

A library of scFv antibodies were screened toward specific MP targets using fluorescent activated cell sorting (FACS). Antibody variants with improved binding to MMP-9 or ADAM-17 showed improved binding and expression to MP targets compared to MMP-9 antibodies in the market such as REGA by an order of magnitude. Next generation and sanger DNA sequencing combined with machine learning approaches revealed the importance of amino acid charges and the length of CDRH3 in these engineered antibodies to bind to specific MP targets. The structure of these antibody variants was studied using computational studies such as molecular dynamic (MD) simulation to elucidate the molecular mechanism of binding to the catalytic site of the target proteases. The protein engineering and protein design techniques developed in these studies could be used for engineering and design of protein binders specifically enzyme inhibitors. The engineered highly selective MP inhibitors has a great potential in developing highly efficient therapeutics with higher efficacy and lower side effects in MP-related diseases. The hybrid TIMP scaffolds developed in these studies offer multi-inhibitory properties critical to address multi-dimensional biomarkers in complex diseases such as cancer which MPs play a key role.