C Kizlik-Masson1, C Vayne1,2, S E McKenzie3, A Poupon4, Y Zhou3, G Champier5, C Pouplard1,2, Y Gruel1,2, J Rollin1,2. 1. UMR CNRS 7292 and Université François Rabelais, Tours, France. 2. Laboratoire d'Hématologie-Hémostase, CHU Tours, Tours, France. 3. Department of Medicine, Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA, USA. 4. PRC, INRA, CNRS, IFCE, Université de Tours, Nouzilly, France. 5. B Cell Design, Limoges, France.
Abstract
Essentials No humanized monoclonal antibody was available to study heparin-induced thrombocytopenia (HIT). We developed the first anti-platelet factor 4 (PF4)/heparin antibody with a human Fc fragment. This antibody (5B9) fully mimics the effects of human HIT antibodies. 5B9 binds two regions within PF4 that may be critical for the pathogenicity of HIT antibodies. SUMMARY: Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of human HIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing human PF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on VH and VL sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.
Essentials No humanized monoclonal antibody was available to study heparin-induced thrombocytopenia (HIT). We developed the first anti-platelet factor 4 (PF4)/heparin antibody with a human Fc fragment. This antibody (5B9) fully mimics the effects of humanHIT antibodies. 5B9 binds two regions within PF4 that may be critical for the pathogenicity of HIT antibodies. SUMMARY: Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of humanHIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing humanPF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on VH and VL sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.