The diversity of the immune response to the A2 domain of human factor VIII.
Approximately 30% of patients with severe hemophilia A develop inhibitory anti-factor VIII (fVIII) antibodies (Abs). We characterized 29 anti-human A2 monoclonal Abs (mAbs) produced in a murine hemophilia A model. A basis set of nonoverlapping mAbs was defined by competition enzyme-linked immunosorbent assay, producing 5 major groups. The overlapping epitopes covered nearly the entire A2 surface when mapped by homolog-scanning mutagenesis. Most group A mAbs recognized a previously described epitope bounded by Arg484-Ile508 in the N-terminal A2 subdomain, resulting in binding to activated fVIII and noncompetitive inhibition of the intrinsic fXase complex. Group B and C mAbs displayed little or no inhibitory activity. Group D and E mAbs recognized epitopes in the C-terminal A2 subdomain. A subset of group D mAbs inhibited the activation of fVIII by interfering with thrombin-catalyzed cleavage at Arg372 at the A1-A2 domain junction. Other group D mAbs displayed indeterminate or no inhibitory activity despite inhibiting cleavage at Arg740 at the A2-B domain junction. Group E mAbs inhibited fVIII light-chain cleavage at Arg1689. Inhibition of cleavages at Arg372 and Arg1689 represent novel mechanisms of inhibitor function and, along with the extensive epitope spectrum identified in this study, reveal hitherto unrecognized complexity in the immune response to fVIII.
Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University, Atlanta, GA, USA.
Rebecca C
RC
Markovitz
John F
JF
Healey
Ernest T
ET
Parker
Shannon L
SL
Meeks
Pete
P
Lollar
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D000918:Antibody Specificity / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006467:Hemophilia A / Q000188:drug therapy / Q000276:immunology*; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013552:Swine; D013917:Thrombin / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005169:Factor VIII; D013917:Thrombin