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D.P. Hart et al.
ed with predicted binding 15-mers. Hence the question of predicted novelty hinged on the position of R593 within the 9-mer: whether at a position involved in MHC-bind- ing (and hence invisible to a TCR), or at a TCR-facing position. As the binding pockets in the MHC groove for HLA-DRB1*01:01 are at positions 1, 4, 6 and 9, both IQR- FLPNPA (with R593 at TCR-facing position 3) and YLTENIQRF (with R593 at TCR-facing position 8) are associated with the formation of pMHC surfaces that are novel in comparison to those formed by endogenous FVIII, as shown in Figure 3B.
Based on the preceding analysis alone, we would pre- dict that a patient with the R593C mutation and HLA-DR allele HLA-DRB1*01:01 would be at risk of developing inhibitors. However, a tFVIII-derived pMHC surface that is novel with respect to an individual’s endogenous FVIII may not be novel in the wider context of his proteome. To evaluate this possibility of a proteome cross-match that reduces the risk of inhibitor development, we searched for a pattern for each of the 9-mer cores matching at T-cell facing positions 2, 3, 5, 7 and 8. For IQRFLPNPA and YLTENIQRF these patterns are XQRXLXNPX and XLTXNXQRX, respectively, where the letter X matches any amino-acid type. These patterns were scanned against a library containing all 11,272,502 unique 9-mers from the human proteome. In this case, the pattern XQRXLXNPX matched the 9-mer FQRELNNPL in human tubulin poly- glutamylase (UniProt25 Q6ZT98), and pattern XLTXNX- QRX matched both the 9-mer GLTENSQRD in dystro- brevin binding protein 1 (dysbindin) (UniProt D6RJC6) and the 9-mer ELTKNAQRA in the uncharacterized
A
B
human protein C2orf48 (UniProt Q96LS8), as shown in Figure 4A.
The final step was to check whether a given cross- matching, proteome-derived 9-mer occurred as a binding core for HLA-DRB1*01:01, as only then would we hypothesize tolerance. In this case, NetMHCII predicted that both FQRELNNPL in tubulin polyglutamylase and ELTKNAQRA in C2orf48 form cores within 15-mers with a predicted IC50 <1000 nmol/L, as shown in Figure 4B. Hence, we ultimately predicted that the F8 missense mutation/HLA allele combination R593C/HLA- DRB1*01:01 confers no, or negligible, risk of inhibitor for- mation owing to fortuitous cross-matches to peptides in the human proteome.
Proteome cross-matches and inhibitor risk stratification
In terms of individual combinations of F8 missense mutation and HLA-DR/DP/DQ isoforms, the impact of proteome cross-matches on predicted risk is shown in a comprehensive heat map (Online Supplementary Figure S1), with a subset of combinations shown in Figure 5. Each individual F8 missense mutation/HLA isoform combina- tion is shown as a single square. An analysis of the full set of data indicates that the percentage of F8 missense muta- tion/HLA isoform combinations associated with predicted inhibitor risk falls appreciably when proteome cross- matches are taken into account: from 49% to 31% with a binding threshold of 1000 nM; and from 37% to 21% with a binding threshold of 500 nM.
These predictions strongly suggest that the risk of
Figure 4. A proteome cross- matching example: Arg593Cys for HLA-DRB1*0101. This exam- ple concerns the two tFVIII binding cores from Figure 3, IQRFLPNPA and YLTENIQRF. (A) Both cores cross-match to 9-mers from pro- teins in the human proteome at TCR-facing positions 2, 3, 5, 7 and 9. (B) Both of the matched 9-mers (one each from tubulin polyglu- tamylase and C2orf48, but none from dysbindin) are predicted by NetMHCII to form binding cores within 15-mers derived from these proteins. Hence, we con- clude that the F8 missense muta- tion/HLA isoform combination Arg593Cys/HLA-DRB1*0101 is associated with negligible risk of inhibitor formation. FVIII: factor VIII; tFVIII: therapeutic factor VIII; TCR: T-cell receptor; HLA: human leukocyte antigen.
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haematologica | 2019; 104(3)