Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9.

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.

Functional label-free assays for characterizing the in vitro mechanism of action of small molecule modulators of capsid assembly.

HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function.

Preclinical evaluation of an anti-alpha5beta1 integrin antibody as a novel anti-angiogenic agent.

Integrin alpha5beta1, the principal fibronectin receptor, is an important survival factor, playing a key role in angiogenesis. Angiogenesis is critical for tumor growth, and anti-angiogenic therapies have met clinical success. To validate the therapeutic potential of an anti-alpha5beta1 strategy, we generated volociximab (M200) a chimeric human IgG4 version of the alpha5beta1 function-blocking murine antibody IIA1; and F200, the Fab derivative. Volociximab, F200 and IIA1 showed similar activity by ELISA (EC50= 0.2nM), Biacore (Kd= 0.1-0.4nM) and inhibition of fibronectin binding (IC50= 2-3nM). The inhibitory potential of alpha5beta1 antibodies was compared to HuMV833, an anti-VEGF antibody. Both volociximab and HuMV833 inhibited HUVEC proliferation (IC50 of volociximab = 0.2-0.5nM; IC50 of HuMV833 = 45nM). However, IIA1, volociximab and F200 were also potent inhibitors of an in vitro model of angiogenesis (HUVEC tube formation assay), unlike HuMV833. Additionally, volociximab inhibited in vitro tube formation induced by VEGF and/or bFGF, suggesting a mechanism of action independent of growth factor stimulus. In fact, inhibition of alpha5beta1 function by volociximab induced apoptosis of actively proliferating, but not resting, endothelial cells. Volociximab does not cross-react with rodent alpha5beta1, therefore in vivo validation of an anti-alpha5beta1 approach was conducted in a cynomolgus model of choroidal revascularization. Volociximab and F200 were potent inhibitors of neovessel formation in this model. These data demonstrate that volociximab has therapeutic potential in diseases in which new vessel formation is a component of the pathology.