HLA DR allele binding analysis of novel mutations and junctions in construct 3 to identify potential neoepitopes. (A) Construct 3, rFVIIIFc-VWF-XTEN, contains an R1648A mutation in the FVIII chain, introduced to avoid cleavage at R1648 and prevent intracellular processing into heavy and light chains. In the D3 domain, amino acids Cys1099 and Cys1142 were mutated to Ala to prevent DʹD3 dimerization. (B,C) Amino acid binding predictions to various HLA DR alleles were evaluated with the NetMHCIIpan, version 3.0, method, as described previously.⁶¹  Binding predictions are depicted before (B; red box; top) and after (B; red box, bottom) mutating WT-R1648 into R1648A in construct 3. After deleting the 9 amino acid residues from the FVIII B-domain linker that captures R1648A (C; green box, top), and before (C; red box; top; green box, bottom) deleting the GAP residues at the FVIII-XTEN junction. GAP residues were from XhoI restriction enzyme site, originally introduced to allow XTEN insertion into FVIII during cloning. IC₅₀, half-maximal inhibitory concentration.