title: Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from a combinatorial phage display library.
abstract: Glycoprotein VI is a type I membrane protein identified as a key platelet receptor for collagen. In vitro binding of the GPVI receptor with collagen leads to activation and ultimately to aggregation of platelets. In vivo, GPVI-collagen interactions could cause formation of occlusive thrombi within vessels with damaged endothelial barriers. GPVI antagonists are therefore important therapeutics in patients suffering from collagen-mediated ischemic disorders such as myocardial infarction or stroke. Polyclonal antibodies to GPVI prepared from one patient serum have previously been described. However, only their monovalent Fab fragments, incapable of receptor crosslinking, were found to protect platelets from collagen-mediated aggregation. Here we describe GPVI-neutralizing human antibodies derived from a combinatorial phage display library of single-chain antibodies. By selecting phage on GPVI-expressing U937 cells, we isolated five specific antibodies - A4, A9, A10, C3 and C9. Of the set A10 and C3 specifically blocked GPVI binding to collagen-rich adventitial layers in aorta sections. The higher affinity antibody A10 inhibited binding of snake-venom convulxin to GPVI. It also specifically protected human platelets from collagen-induced aggregation in vitro. A10-bound platelets could still be activated by ADP or thrombin suggesting that this human scFv may represent an original anti-platelet agent for the treatment of collagen-mediated thrombotic diseases.
authors.affiliation: Millennium Pharmaceuticals, Inc, 640 Memorial Drive, Cambridge, MA 02139, USA.
authors.forename: Ming D
authors.initials: MD
authors.lastname: Qian
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Villeval
authors.forename: Ximing
authors.initials: X
authors.lastname: Xiong
authors.forename: Martine
authors.initials: M
authors.lastname: Jandrot-Perrus
authors.forename: Kumiko
authors.initials: K
authors.lastname: Nagashima
authors.forename: James
authors.initials: J
authors.lastname: Tonra
authors.forename: Kevin
authors.initials: K
authors.lastname: McDonald
authors.forename: Andrew
authors.initials: A
authors.lastname: Goodearl
authors.forename: Davinder
authors.initials: D
authors.lastname: Gill
mesh_terms: D000818:Animals; D018955:CD36 Antigens / Q000276:immunology*; D003094:Collagen / Q000494:pharmacology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000494:pharmacology*; D051379:Mice; D009500:Neutralization Tests; D019151:Peptide Library; D010974:Platelet Aggregation / Q000187:drug effects*; D020298:U937 Cells
chemical_list: D018955:CD36 Antigens; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C055966:immunoglobulin Fv; D003094:Collagen