Site-specific targeting of antibody activity in vivo mediated by disease-associated proteases
ABSTRACT As a general strategy to selectively target antibody activity in vivo, a molecular architecture was designed to render binding activity dependent upon proteases in disease tissues. A protease-activated antibody (pro-antibody) targeting vascular cell adhesion molecule 1 (VCAM-1), a marker of atherosclerotic plaques, was constructed by tethering a binding site-masking peptide to the antibody via a matrix metalloprotease (MMP) susceptible linker. Pro-antibody activation in vitro by MMP-1 yielded a 200-fold increase in binding affinity and restored anti-VCAM-1 binding in tissue sections from ApoE⁻/⁻ mice ex vivo. The pro-antibody was efficiently activated by native proteases in aorta tissue extracts from ApoE⁻/⁻, but not from normal mice, and accumulated in aortic plaques in vivo with enhanced selectivity when compared to the unmodified antibody. Pro-antibody accumulation in aortic plaques was MMP-dependent, and significantly inhibited by a broad-spectrum MMP inhibitor. These results demonstrate that the activity of disease-associated proteases can be exploited to site-specifically target antibody activity in vivo.
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ABSTRACT: This review presents a brief overview of the general categories of commercially used proteases, and critically surveys the successful strategies currently being used to improve the properties of proteases for various commercial purposes. We describe the broad application of proteases in laundry detergents, food processing, and the leather industry. The review also introduces the expanding development of proteases as a class of therapeutic agents, as well as highlighting recent progress in the field of protease engineering. The potential commercial applications of proteases are rapidly growing as recent technological advances are producing proteases with novel properties and substrate specificities.FEBS letters 01/2013; 587(8). DOI:10.1016/j.febslet.2012.12.019 · 3.34 Impact Factor
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ABSTRACT: Based on the powerful cell-penetrating ability of low molecular weight protamine (LMWP) and the over-expression of matrix metalloproteinases in the tumor sites, we here constructed an activatable low molecular weight protamine (ALMWP) and modified it onto the surface of PEG-PLA nanoparticles to develop a 'smart' drug delivery system with enhanced permeability for facilitating site-specific targeting delivery of anticancer drug. The obtained ALMWP-NP with a particle size of 134.0 ± 4.59 nm and a zeta potential of -34.4 ± 2.7 mV, exhibited an enhanced MMP-dependent accumulation in HT-1080 cells via both energy-independent direct translocation and clathrin-mediated, cytoskeleton-dependent endocytosis. Pharmacokinetic and biodistribution study in HT-1080 tumor-bearing mice showed that ALMWP-NP significantly increased the accumulation of paclitaxel (PTX) in the tumor site but not the non-target tissues. In addition, intra-tumor distribution analysis demonstrated that more ALMWP-NP penetrated deeply into the tumor parenchyma. As a result, PTX loaded by ALMWP-NP exhibited improved anti-tumor efficacy over that by NP and LMWP-NP. The findings suggested that ALMWP-NP could be used as a safe and effective tumor-targeting drug delivery system and opened a new gateway to the application of cell-penetrating peptides for targeted anti-tumor therapy.Bioconjugate Chemistry 01/2013; 24(3). DOI:10.1021/bc300520t · 4.82 Impact Factor
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ABSTRACT: Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.Science translational medicine 10/2013; 5(207):207ra144. DOI:10.1126/scitranslmed.3006682 · 14.41 Impact Factor