title: Six amino acid residues in a 1200 Å2 interface mediate binding of factor VIII to an IgG4κ inhibitory antibody.
abstract: The development of neutralizing anti-factor VIII (FVIII) antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF) and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200) plus two non-contiguous arginines (FVIII-R2215 and R2220), contributed appreciably to the affinity. B-domain-deleted (BDD) FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies.
authors.affiliation: Puget Sound Blood Center Research Institute, Seattle, Washington, United States of America; Division of Hematology, Dept. Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Jasper C
authors.initials: JC
authors.lastname: Lin
authors.affiliation: Puget Sound Blood Center Research Institute, Seattle, Washington, United States of America.
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Ettinger
authors.affiliation: General Electric (GE) Healthcare Life Sciences, Portland, Oregon, United States of America.
authors.forename: Jason T
authors.initials: JT
authors.lastname: Schuman
authors.affiliation: Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
authors.forename: Ai-Hong
authors.initials: AH
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Walter Reed National Military Medical Center, Bethesda, Maryland, United States of America.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Wamiq-Adhami
authors.affiliation: Puget Sound Blood Center Research Institute, Seattle, Washington, United States of America.
authors.forename: Phuong-Cac T
authors.initials: PC
authors.lastname: Nguyen
authors.affiliation: Puget Sound Blood Center Research Institute, Seattle, Washington, United States of America.
authors.forename: Shelley M
authors.initials: SM
authors.lastname: Nakaya-Fletcher
authors.affiliation: Puget Sound Blood Center Research Institute, Seattle, Washington, United States of America.
authors.forename: Komal
authors.initials: K
authors.lastname: Puranik
authors.affiliation: Puget Sound Blood Center Research Institute, Seattle, Washington, United States of America; Division of Hematology, Dept. Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Thompson
authors.affiliation: Puget Sound Blood Center Research Institute, Seattle, Washington, United States of America; Division of Hematology, Dept. Medicine, University of Washington, Seattle, Washington, United States of America; Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
authors.forename: Kathleen P
authors.initials: KP
authors.lastname: Pratt
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry*; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D008958:Models, Molecular; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D014841:von Willebrand Factor; D005169:Factor VIII