Abstract
Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the β-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits β-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive β-tryptase monomers, and may provide an alternative strategy for antibody engineering.
Highlights
Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma
The activated tetramer stimulates a range of inflammatory and tissue remodeling processes through cleavage of a variety of substrates including proteinase-activated receptor (PAR-2), vasoactive intestinal peptide (VIP), matrix metalloproteinases (MMPs), fibronectin, and collagen[7,8,9,14]
Given that the active site of β-tryptase is not readily accessible to macromolecules and that rabbit antibodies possess long complementarity-determining region (CDR) that might enable additional MOAs, we generated a panel of antitryptase antibodies via immunization of rabbits followed by standard hybridoma technology[32]
Summary
Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Activation of mast cells induces the release of secretory granules that contain histamine, cytokines, chemokines as well as high levels of various proteases[3,5], in particular the trypsin-like serine protease tryptase, which is present in very high concentrations[6]. This burst of protease activity leads to many consequences such as increased collagen production by lung fibroblasts and proliferation and contraction of lung smooth muscle cells[4,7,8,9]. The only known natural macromolecular inhibitors of β-tryptases are leech-derived tryptase inhibitor (LDTI), tick-derived protease inhibitor (TdPI), and lactoferrin[21,22,23]
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