Routine measurements of factor VIII activity and inhibitor titer in the presence of emicizumab utilizing anti-idiotype monoclonal antibodies

J Thromb Haemost. 2018 Jul;16(7):1383-1390. doi: 10.1111/jth.14135. Epub 2018 May 27.

Abstract

Essentials Emicizumab (Emi) affects the APTT-based assays of factor (F)VIII activity and inhibitor titer. A mixture of two anti-Emi monoclonal antibodies (mAb) effectively neutralized the Emi activity. Anti-Emi mAbs completely eliminated the influence of Emi on FVIII activity and inhibitor titer. The inclusion of anti-Emi mAbs in routine FVIII assays would be useful for Emi-treated patients.

Summary: Background Emicizumab is an anti-factor (F)IXa/X bispecific monoclonal antibody (mAb), mimicking the factor (F)VIIIa cofactor activity. Emicizumab does not require activation by thrombin and its shortening effect on the activated partial prothrombin time (APTT) is more pronounced than that of factor (F)VIII. APTT-based FVIII activity (FVIII:C) and FVIII inhibiter titer measurements are influenced by the presence of emicizumab. Aim To establish a reliable APTT-based assay to measure FVIII in the presence of emicizumab. Methods Plasmas from hemophilia A (HA) patients without or with inhibitors were studied using one-stage FVIII:C and Bethesda inhibitor assays. Two recombinant anti-idiotype mAbs to emicizumab (anti-emicizumab mAbs) were prepared, rcAQ8 to anti-FIXa-Fab and rcAJ540 to anti-FX-Fab. Results The combined anti-idiotype mAbs (2000 nm each) eliminated the effects of emicizumab on APTTs of HA plasmas without or with inhibitor by competitive inhibition of antibody binding to FIX(a)/FX(a). Measurements of FVIII coagulation activity in HA plasmas without inhibitor were overestimated in the presence of emicizumab (1 μm = ~150 μg mL-1 ) at all reference levels of FVIII. The addition of anti-emicizumab mAbs to the assay mixtures completely neutralized the emicizumab and facilitated accurate determination of FVIII:C. Anti-FVIII inhibitor titers were undetectable in the presence of emicizumab in HA plasmas with inhibitor or normal plasmas mixed with anti-FVIII neutralizing antibodies. These effects of emicizumab were completely counteracted by the addition of the anti-idiotype mAbs, allowing accurate assessment of inhibitor titers. Conclusion The in vitro inclusion of anti-emicizumab mAbs in the standard one-stage coagulation assays prevented interference by emicizumab and enabled accurate measurements of FVIII:C and inhibitor titers.

Keywords: anti-idiotypic antibody; blood coagulation factor inhibitor; emicizumab; factor VIII; hemophilia A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Bispecific / blood
  • Antibodies, Bispecific / immunology
  • Antibodies, Bispecific / pharmacology*
  • Antibodies, Monoclonal, Humanized / blood
  • Antibodies, Monoclonal, Humanized / immunology
  • Antibodies, Monoclonal, Humanized / pharmacology*
  • Antibodies, Neutralizing / blood*
  • Antibodies, Neutralizing / immunology
  • Binding, Competitive
  • Blood Coagulation / drug effects*
  • Coagulants / blood
  • Coagulants / immunology
  • Coagulants / pharmacology*
  • Dose-Response Relationship, Drug
  • Factor IXa / immunology
  • Factor IXa / metabolism
  • Factor VIII / analysis*
  • Factor VIII / immunology
  • Factor Xa / immunology
  • Factor Xa / metabolism
  • Hemophilia A / blood*
  • Hemophilia A / diagnosis
  • Hemophilia A / immunology
  • Humans
  • Partial Thromboplastin Time*
  • Predictive Value of Tests
  • Protein Binding
  • Reproducibility of Results

Substances

  • Antibodies, Bispecific
  • Antibodies, Monoclonal, Humanized
  • Antibodies, Neutralizing
  • Coagulants
  • emicizumab
  • F8 protein, human
  • Factor VIII
  • Factor IXa
  • Factor Xa