Fc modification to transform a pathogenic antibody into a potential therapeutic for thromboinflammatory disease

Abstract

Abstract KKO is a HIT (heparin-induced thrombocytopenia)-like mouse monoclonal antibody (moAb) that binds to platelet factor 4 (PF4) when complexed to polyanions like heparin and neutrophil extracellular traps (NETs). Human (h) PF4 protected NETs from DNases and was further enhanced by KKO as it activates platelets and neutrophils via both FcgRIIA and complement, causing prothrombotic state in transgenic mice expressing FcgRIIA and hPF4. We posited through that if Fc activity of KKO were eliminated then it may stabilize NETs and potentially be therapeutic by stabilizing NETs without FcgRIIA and complement activation. We deglycosylated Fc region of KKO to make DGKKO, and tested if we can avoid detrimental effects without interfering with KKO protection of PF4:NET complexes. We also speculated that DGKKO may bind to hPF4:polyanions and prevent them from inducing HIT. Binding efficacy of DGKKO to PF4-heparin complexes was studied by ELISA and dynamic light scattering. Platelet and complement activation were also studied for DGKKO, and we found that DGKKO still bind to PF4-polyanion complexes, with no platelet activation and significantly lower complement activation. DGKKO also enhanced NET nuclease resistance to improve microbial capture by PF4-NETs, preventing thrombocytopenia and improving survival in murine sepsis models. Also, our humanized in vitro microfluidics studies as well as HIT mice studies showed that infusion of DGKKO prevented both thrombocytopenia and thrombosis in a murine model of HIT. These results showed that Fc-modified KKO has protective effects in at least two models of thromboinflammation. Further studies are underway to examine the role of NET stabilization by modified KKO in other thromboinflammatory disorders. R35 to Mortimer Poncz and CHOP Foerderer Grant to Amrita Sarkar