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 non-pathogenic 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 monoclonal antibody (KKO) that recognizes a binding site overlapping with pathogenic HIT patient antibodies on PF4. We developed a wildtype 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 following phage biopanning. We confirmed high nanomolar affinity and specific binding of these scFv fragments to PF4/heparin complexes using anti-PF4/heparin enzyme immunoassays (EIAs) and biolayer-interferometry (BLI) and demonstrated that KKO and all scFvs bind to an overlapping heparin-dependent epitope on PF4. 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 (SRA). Importantly, the binding of non-pathogenic 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.