OC 41.4 - Patient anti-FVIII drug antibodies bind preferentially to a subset of FVIII covalent forms

Background: Haemophilia A is a chronic life-threatening condition caused by deficiency or dysfunction of plasma coagulation factor VIII (FVIII). Prophylaxis by regular intravenous infusion of exogenously derived FVIII concentrates is a common treatment. A major obstacle to FVIII replacement therapy is the generation of alloantibodies (inhibitors) that diminish efficacy. Approximately one-third of severe haemophilia A patients, and a smaller fraction of mild or moderate patients, develop neutralising anti-FVIII antibodies that render the drug non-functional.

Aims: To determine the disulfide-bonded states of native and recombinant FVIII and their contribution to FVIII anti-drug antibody development.

Methods: Disulfide-bonded states of plasma and recombinant FVIII were quantified using differential cysteine alkylation and mass spectrometry. A panel of 4 patient-derived FVIII-neutralising antibodies were coupled to magnetic beads, incubated with recombinant FVIII and the disulfide-bonded states of the bound and free FVIII determined.

Results: All 8 structurally defined FVIII disulfide bonds were found to exist in unformed states in different proportions of plasma FVIII molecules. The bonds were unformed in 1 in 10 (C172-C198 in the A1 domain) to 1 in 2 (C1918-C1922 in the A3 domain) molecules of the FVIII populations. The different disulfide-bonded states of FVIII were predetermined and not a result of post-secretion redox events in the blood as recombinant FVIII had the same disulfide status as the plasma protein. The 4 patient anti-FVIII antibodies bound preferentially to FVIII covalent forms where 2 or 3 of the 8 disulfides are significantly (p < 0.01) more unformed; C1918-C1922 in the A3 domain, C2040-C2188 in the C1 domain and C2193-C2345 in the C2 domain.

Conclusion(s): FVIII is constitutively made as multiple partially disulfide-bonded forms and there is a relationship between FVIII immunogenicity and the state of the disulfide bonds. These findings imply that it may be possible to engineer a FVIII that diminishes the emergence of inhibitors.