Function-modulating human monoclonal antibodies against platelet-membrane receptors isolated from a phage-display library.
Monoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbalpha binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies.
Hagay
Y
Y
Bio-Technology General Inc., Rehovot, Israel. yochih@btgil.com
Lahav
J
J
Levanon
A
A
Panet
A
A
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005343:Fibrinolytic Agents / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000494:pharmacology; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014841:von Willebrand Factor / Q000737:chemistry
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; C023592:glycocalicin; D014841:von Willebrand Factor