Factor VIII (FVIII) neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 (legacy FVIII numbering, sequence: SYFTNMFATWSPSKARLHLQ) has been shown to contain an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells isolated from HA subjects. The aim of this study was to identify amino acid substitutions to de-immunize this epitope while retaining pro-coagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The minimal DRB1*01:01-binding motif was mapped to FVIII2194-2205 by quantitative peptide-MHC Class II (MHCII) binding assays. Further binding assays with sequence-modified FVIII peptides established that the residues important for DRB1*01:01 affinity were F2196, M2199, A2201, and S2204, corresponding to MHCII anchor positions 1, 4, 6 and 9. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation and cytokine secretion of five clones specific for FVIII2194-2213. The substitution F2196A was then engineered into a recombinant (r)FVIII-C2 protein. T-cell clones that were strongly stimulated by wild-type rFVIII-C2 did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic synthetic peptide includes a naturally processed T-cell epitope. The measured and predicted effects of additional amino acid substitutions within this binding motif were further evaluated by peptide-MHCII binding assays, CD4+ T-cell proliferation assays, and MHCII-binding prediction algorithms. Amino acid homologs within this binding motif were evaluated with sequence homologies to FVIII and FV proteins from different species. On the basis of all these results, six rB-domain-deleted (BDD)-FVIII proteins with amino acid substitutions F2196A, F2196L, F2196K, M2199A, M2199W or M2199R, respectively, were designed and produced; all but the F2196A variant had specific activities comparable to that of rWT-BDD-FVIII. The F2196A variant also had the lowest expression level among the rBDD-FVIIII proteins produced. Peptide-MHCII binding assays confirmed that FVIII2194-2205 peptides with substitutions F2196A, F2196K, M2199A and M2199R did not bind to DRB1*01:01 or to an additional 9 HLA-DR proteins tested, with the exception of the M2199A peptide binding to DRB1*09:01. The predicted effects of these FVIII sequence modifications on binding to additional MHCII alleles included in reference sets representing ~99% of the human population HLA diversity were evaluated using ProPred and Immune Epitope Database algorithms. These programs correctly predicted that the substitutions would abrogate binding to DRB1*01:01 and also indicated that affinities for additional MHCII would be reduced by each substitution. The substitutions were predicted to increase peptide-MHCII affinities for a smaller number of MHCII alleles, indicating a possible risk of creating neo-epitopes restricted to these specific HLA alleles. Importantly, proliferation of patient-derived T-cell clones in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A proteins was dramatically reduced compared with responses to rWT-BDD-FVIII. We have previously demonstrated that a severe HA inhibitor subject had an oligoclonal T-cell response to only one T-cell epitope within the FVIII A2, C1 and C2 domains and that one epitope was FVIII2194-2213 (Ettinger et al., Blood 2106; 128(16):2043). This observation, together with the results presented here, further suggests that modification of only a small number of immunodominant epitopes may be sufficient to significantly de-immunize FVIII and decrease FVIII inhibitors in HA patients. The present results provide proof-of-principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients.
Ettinger: Bloodworks NW: Patents & Royalties: Inventor on FVIII patents. James: Bloodworks NW: Patents & Royalties: inventor on FVIII patent(s). Pratt: Bloodworks NW: Patents & Royalties: inventor on FVIII patents.
Asterisk with author names denotes non-ASH members.