Engineering single-chain variable fragments to identify pathogenic antibodies in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is characterized by the production of pathogenic antibodies that bind to complexes of platelet factor 4 (PF4) and heparin, causing platelet activation and a hypercoagulable state. A significant proportion of heparin-treated patients develop nonpathogenic anti-PF4/heparin antibodies that do not activate platelets but interfere with routine screening tests causing frequent false-positive results. We recently showed that pathogenic HIT antibodies are monoclonal and bind to an overlapping PF4 binding site, which could be exploited to identify clinically significant antibodies in patients. This study aimed to develop epitope-specific inhibitors based on the HIT-like murine monoclonal antibody (KKO) that recognizes a binding site overlapping with patient-derived pathogenic HIT antibodies on PF4. We developed a wild-type single-chain variable fragment (scFv) derived from KKO and performed site-directed mutagenesis to create a library of mutant anti-PF4/heparin scFv sequences. Five candidate scFvs were selected based on sequence enrichment after phage biopanning. We confirmed the high affinity and specific binding of these scFv fragments to PF4/heparin complexes using enzyme immunoassays (EIAs) and biolayer interferometry and demonstrated that KKO and all scFvs bind to an overlapping heparin-dependent site on PF4 by epitope mapping. Using patient sera, we demonstrated that our scFv candidates selectively inhibit the binding of pathogenic anti-PF4/heparin HIT antibodies and prevented platelet activation of some samples in the serotonin release assay. Importantly, the binding of nonpathogenic antibodies to PF4/heparin was unperturbed. These findings support the use of scFvs targeting a shared pathogenic region on PF4 to improve the diagnostic accuracy of anti-PF4/heparin EIAs for HIT.