title: Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz-type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor.
abstract: BACKGROUND
  A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab.
  
  
  METHODS
  Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats.
  
  
  CONCLUSIONS
  Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism.
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark. Electronic address: LeeH@novonordisk.com.
authors.forename: Lene
authors.initials: L
authors.lastname: Hansen
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Lars Christian
authors.initials: LC
authors.lastname: Petersen
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Brian
authors.initials: B
authors.lastname: Lauritzen
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Jes Thorn
authors.initials: JT
authors.lastname: Clausen
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Susanne Nedergaard
authors.initials: SN
authors.lastname: Grell
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Agersø
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Brit Binow
authors.initials: BB
authors.lastname: Sørensen
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Ida
authors.initials: I
authors.lastname: Hilden
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Kasper
authors.initials: K
authors.lastname: Almholt
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology / Q000493:pharmacokinetics*; D001777:Blood Coagulation; D005260:Female; D006801:Humans; D008074:Lipoproteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D051379:Mice; D011480:Protease Inhibitors / Q000276:immunology / Q000493:pharmacokinetics*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D014018:Tissue Distribution; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000276:immunology
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D008074:Lipoproteins; D011480:Protease Inhibitors; C051928:lipoprotein-associated coagulation inhibitor; C574488:concizumab; D014360:Trypsin Inhibitor, Kunitz Soybean
keywords: Antibody; Extrinsic pathway; Immunofluorescence; Pharmacokinetics; TFPI; Tissue factor pathway inhibitor