Generation of Protease-Inhibiting Monoclonal Antibodies By Novel Paratope Design and Function-Based Screening

by novel paratope design and function-based screening

Dong Hyun Nam, Tyler Lopez, Kuili Fang, Xin Ge*
Chemical and Environmental Engineering, University of California, Riverside
900 University Ave, Riverside, CA 92521

As extremely important signaling molecules, proteases precisely control a wide variety of physiological processes, and thus many diseases are associated with altered protease expression or substrate proteolysis. Consequently, proteases are one of the largest families of pharmaceutical targets, and an apparent pharmaceutical mechanism is to block abnormal or pathogenic proteolysis by inhibiting the catalytic reactions. Most therapeutic protease inhibitors currently in clinical use or under developments are peptides or their chemical compound mimics. Because ~2% of the human genome is estimated to encode proteases with at least 500-600 proteases have been identified, specificity is a crucial property for any protease inhibition therapy. However, achieving target specificity has been recognized as the main hurdle for protease inhibitor developments. This is because the reaction mechanism is highly conserved among the same class or family of proteases, resulting in lead compounds often inhibit multiple targets and potentially causing unwanted side effects. With these aspects, antibody-based inhibitors are emerging as very attractive therapeutic agents due to their exclusive specificity given by a large antigen-antibody contact surface.

Although isolating antigen-specific mAbs has become routine, developing antibodies with protease inhibitory functions is still challenging, because of (1) low antigenicity of the enzymatic active sites, (2) lack of a function-based selection method. This study devolved a panel of novel techniques to address these obstacles and used matrix metalloproteinase (MMP)-14 as the target, which plays important role in tumor growth, invasion and neovascularization.

Inspired by the fact that a large proportion of antibodies isolated from camels and llamas can bind to active pockets and inhibit enzymatic reactions, we analyzed inhibitory antibody sequences and structures, and hypothesized that convex shaped paratopes given by ultra long CDRs are inhibition-prone. Large synthetic human antibody libraries (>10⁹ variants) carrying extended CDR-H3 ranging from 23 to 27 aa were constructed. After phage panning on MMP-14, dozens of Fab clones exhibiting strong binding signals were isolated, with nM range of affinity for many of them. CDR-H3 sequences of 40 randomly picked binding clones showed dramatic patterns with central histidines flanked by arginine rich sequences. These residues presumably chelate the catalytic zinc and recognize negatively charged surface of the reaction pocket of MMP-14, respectively. More strikingly majority of these sequenced Fabs demonstrated inhibitory activity with various potencies. Regarding to antibody selection method, current technology is essential binding-based assays with little controls on epitope specificity or inhibitory functions. In this study, we developed a function-based high-throughput screening method by achieving epitope-specific FACS that simultaneously performed selection on target protease (MMP-14) and counter-selection on its inhibitor (TIMP-2), which were conjugated with different fluorophores. Able to enrich inhibitory clones from non-specific or non-inhibitory clones, this method was applied for affinity maturation of inhibitory mAbs. In addition, we also applied motif grafting to convert a low affinity peptide inhibitor into a high potent mAb with 2000 folds of improvements in inhibition potency from 150 µM to 70 nM. These novel techniques significantly advanced our understanding of enzymatic inhibition mechanisms and greatly facilitated the discovery of highly selective and highly potent inhibitory antibodies, thus are expected to have a broad range of applications in pharmaceutical and biotechnological industries. Furthermore, because the vulnerable sites for drug binding of many viral coating proteins represent concave conformations, the synthetic antibody libraries with convex paratopes developed in this study also have potentials for the development of neutralizing antibodies.