Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases

Abstract

KLK1 (tissue kallikrein 1) is a member of the tissue kallikrein family of serine proteases and is the primary kinin-generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki=0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface-plasmon-resonance biosensor assay when KLK1 was complexed to known active-site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were each tested at a concentration of 1 microM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial epithelial cells grown at the air/liquid interface, DX-2300 blocked oxidative-stress-induced epidermal-growth-factor receptor activation and downstream mucus cell proliferation and hypersecretion, which have been previously shown to be mediated by KLK1. In an allergic sheep model of asthma, DX-2300 inhibited both allergen-induced late-phase bronchoconstriction and airway hyper-responsiveness to carbachol. These studies demonstrate that DX-2300 is a potent and specific inhibitor of KLK1 that is efficacious in in vitro and in vivo models of airway disease.

Unleashing the therapeutic potential of human kallikrein-related serine proteases

“…The therapeutic usefulness of KLK-based antibodies has also been reported. Examples include the successful use of the fully humanized KLK1-specific antibody DX-2300 in reducing late-phase allergen-induced bronchoconstriction and airway hypersensitivity in a sheep model of asthma, and the use of KLK6-neutralizing antibodies in reducing the severity of symptoms in experimental autoimmune encephalomyelitis and in a viral mouse model of multiple sclerosis 136,175,176 .…”

“…Consistent with this pathway, in vivo inhalation or intravenous administration of a KLK1-specific antibody known as DX-2300 (Dyax) in a sheep model of allergic asthma resulted in a substantial inhibition of late-phase allergen-induced bronchoconstriction and of airway hyperresponsiveness 136 . DX-2300 is a fully humanized antibody developed through phage-display technologies to specifically target KLK1 through a competitive inhibition mechanism, with an inhibitory constant (K i ) of 0.13 nM 136 .…”

10 Kallikrein-Kinin Cascade: Bioregulation by Human Tissue Kallikrein 1 (hK1, KLK1)

“…DX-2300 acts via a competitive inhibition mechanism (K i = 0.13 nM) by interacting with the active site triad of Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 7/26/15 3:44 PM hK1. This study proved that DX-2300 is a potent and specific inhibitor of hK1, which is efficacious in in vitro and in vivo models of airway disease (Sexton et al, 2009). …”

“…The importance of hK1 in asthmatic subjects is highlighted by the fact that DX-2300, a human antibody that inhibits hK1 via a competitive inhibition mechanism, blocks the oxidative stress induced by epidermal growth factor receptor activation and downstream mucous cell proliferation and hypersecretion. Furthermore, in the allergic sheep model of asthma, DX-2300 inhibits both allergen-induced late-phase bronchoconstriction and airway hyperresponsiveness to carbachol (carbamylcholine, a cholinomimetic drug that activates the acetylcholine receptor (Sexton et al, 2009). (brown).…”

Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

“…Purified Fab product was exchanged into a buffer containing 50 m m citrate/phosphate, 100 m m NaCl, pH 6.0, through an ultrafiltration/diafiltration process using a Millipore Amicon Ultra concentration unit (10,000-kDa molecular mass cutoff). Recombinant Fab fragments were reformatted into full-length human IgG1 antibodies (f-allotype) (39), expressed as stable transfections in CHO cells using a fed-batch fermentation strategy, and purified as previously described (40) into formulation buffer (100 m m citrate/phosphate buffer, pH 6.0, 50 m m NaCl, 2% (w/v) trehalose, and 0.01% Tween 80).…”