Single‐chain antibodies for the conformation‐specific blockade of activated platelet integrin α IIb β 3 designed by subtractive selection from naïve human phage libraries

Binding of fibrinogen to platelet integrin α IIb β 3 mediates platelet aggregation, and thus inhibition of α IIb β 3 represents a powerful therapeutic strategy in cardiovascular medicine. However, the currently used inhibitors of α IIb β 3 demonstrate several adverse effects like thrombocytopenia and bleeding, which are associated with their property to bind to non‐activated α IIb β 3 . To circumvent these problems, we designed blocking single‐chain antibody‐fragments (scFv) that bind to α IIb β 3 exclusively in its activated conformation. Two naïve phage libraries were created: a natural phage library, based on human lymphocyte cDNA, and a synthetic library, with randomized V H CDR3. We performed serial rounds of subtractive panning with depletion on non‐activated and selection on activated α IIb β 3 , which were provided on resting and ADP‐stimulated platelets and CHO cells, expressing wild‐type or mutated and thereby activated α IIb β 3 . In contrast to isolated, immobilized targets, as generally used for phage display, this unique cell‐based approach for panning allowed the preservation of functional integrin conformation. Thereby, we obtained several scFv‐clones that demonstrated exclusive binding to activated platelets and complete inhibition of fibrinogen binding and platelet aggregation. Interestingly, all activation‐specific clones contained an RXD pattern in the HCDR3. Binding studies on transiently expressed point mutants and mouse‐human domain‐switch mutants of α IIb β 3 indicate a binding site similar to fibrinogen. In conclusion, we generated human activation‐specific scFvs against α IIb β 3 , which bind selectively to activated α IIb β 3 and thereby potently inhibit fibrinogen binding to α IIb β 3 and platelet aggregation.