https://orcid.org/0000-0001-7441-4554
Key Points
FVIII is constitutively made as multiple partially disulfide-bonded states.
Patient-derived anti-FVIII antibodies bind preferentially to a subset of FVIII disulfide-bonded states.
Haemophilia A is a chronic life-threatening condition caused by deficiency or dysfunction of plasma coagulation factor VIII (FVIII) and commonly managed by prophylaxis with regular infusion of FVIII protein. A major obstacle to FVIII replacement therapy is the generation of alloantibodies that diminish efficacy. Disulfide bonds link pairs of cysteine residues in proteins and in several proteins have been found to be only partially formed in the mature proteins. FVIII contains 8 disulfide bonds and their redox state in human blood and recombinant FVIII was determined using differential cysteine alkylation and mass spectrometry. All 8 disulfide bonds were found to be unformed in ~10% to ~70% of molecules of FVIII populations, which suggested a conformational flexibility that could favour binding of certain ligands to subsets of FVIII with more or less formed disulfide bonds. To test this hypothesis, the binding of a panel of five patient-derived anti-FVIII antibodies to the population of FVIII disulfide-bonded states was evaluated. All five antibodies bound preferentially to FVIII states where 2 or 3 of the 8 disulfides are significantly more unformed; C1918-C1922 in the A3 domain, C2040-C2188 in the C1 domain and C2193-C2345 in the C2 domain. Disulfide bond mutagenesis experiments and molecular dynamics simulations indicate that this subset of FVIII states have long-range conformational dynamism that favours anti-drug antibody binding. These findings will assist efforts to engineer a FVIII molecule that is less prone to neutralization by anti-drug antibodies and has general implications for autoimmune conditions and antibody drug efficacy.
