Characterization of anti-PF4 monoclonal antibodies to study vaccine-induced immune thrombocytopenia and thrombosis

Adenoviral vector-based vaccines against COVID-19 cause vaccine-induced immune thrombocytopenia and thrombosis (VITT), a rare but severe adverse reaction caused by IgG antibodies against platelet factor 4 (PF4). To study VITT, patient samples are crucial but have become a scarce resource. Recombinant monoclonal antibodies (moAbs) consisting of clonotypic amino acid sequences of anti-PF4 IgG derived from VITT patients are a new alternative to study VITT pathophysiology.

Amino-acid sequences of the variable region of immunoglobulin heavy and light chain of anti-PF4 IgG derived from VITT patients were obtained by mass-spectrometric sequencing and moAbs were recombinantly synthetized in a novel reverse-engineering approach. Eight different moAbs were produced: moAb1a-c (from 1 patient after vaccination with Janssen Johnson&Johnson), moAb2a-b, moAb3 and moAb4a-b (from 3 patients after vaccination with AstraZeneca). These moAbs were further characterized using rapid PF4/polyanion- and PF4-chemiluminescence assays and anti-PF4 ELISA, as well as PF4-induced platelet activation assay (PIPA) using washed platelets.

All moAbs bound to PF4 alone, but not to PF4/polyanion complexes in chemiluminescence assays. Anti-PF4 ELISA (Figure 1A) revealed concentration dependent PF4 binding of moAbs, a similar pattern was detected for platelet activation in the PIPA (Figure 1B). MoAbs derived from different patients varied in their avidities.

MoAbs sharing clonotypic amino-acid sequences of anti-PF4 IgG variable region from VITT patients reflect VITT-typical binding capacities and the ability to activate platelets. Therefore, these moAbs offer an attractive new option to study VITT pathophysiology.

Disclosure statements of the authors can be obtained upon request.