Bloodworks Northwest Research Institute, Seattle, WA.
Benaroya Research Institute, Seattle, WA.
Blood Adv. 2018 Feb 27;2(4):309-322. doi: 10.1182/bloodadvances.2017013482.
Factor VIII (FVIII)-neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII contains an immunodominant HLA-DRA01-DRB101:01 (DRB101:01)-restricted epitope recognized by CD4 T-effector cells from HA subjects. The aim of this study was to identify amino acid substitutions to deimmunize this epitope while retaining procoagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The shortest DRB101:01-binding peptide was FVIII, and residues important for affinity were identified as F2196, M2199, A2201, and S2204. T-cell proliferation experiments with Ala-substituted FVIII peptides identified F2196A as a substitution that abrogated proliferation of clones specific for the WT sequence. T-cell clones that were stimulated by recombinant WT-FVIII-C2 (rWT-FVIII-C2) protein did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic peptide includes a naturally processed T-cell epitope. Additional amino acid substitutions at F2196 and M2199 were evaluated by peptide-MHC class II (MHCII)-binding assays, T-cell proliferation assays, epitope prediction algorithms, and sequence homologies. Six B-domain-deleted (BDD)-FVIII proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W, or M2199R were produced. Proliferation of T-cell clones and polyclonal lines in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A was reduced compared with responses to WT-BDD-FVIII. The BDD-FVIII-F2196K sequence modification appears to be the most promising sequence variant tested here, due to its effectiveness at eliminating DRB1*01:01-restricted immunogenicity, low potential immunogenicity in the context of other MHCII alleles, expression level comparable to WT-BDD-FVIII, and retained procoagulant activity. These results provide proof of principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients.
凝血因子 VIII（FVIII）中和抗体（抑制剂）是 A 型血友病（HA）的严重并发症。该肽段 FVIII 包含一个免疫显性 HLA-DRA01-DRB101:01（DRB101:01）限制性表位，可被 HA 受试者的 CD4 效应 T 细胞识别。本研究的目的是鉴定氨基酸取代，以消除该表位的免疫原性，同时保留与野生型（WT）FVIII 蛋白相当的促凝活性和表达水平。与 DRB101:01 结合最短的肽段是 FVIII，确定对亲和力很重要的残基是 F2196、M2199、A2201 和 S2204。用 Ala 取代 FVIII 肽进行 T 细胞增殖实验，鉴定出 F2196A 取代可消除针对 WT 序列的克隆增殖。用重组 WT-FVIII-C2（rWT-FVIII-C2）蛋白刺激的 T 细胞克隆在与 rFVIII-C2-F2196A 共培养时不增殖，表明免疫原性肽包含一个天然加工的 T 细胞表位。通过肽-MHC 类 II（MHCII）结合测定、T 细胞增殖测定、表位预测算法和序列同源性进一步评估了 F2196 和 M2199 的其他氨基酸取代。生产了具有 F2196A、F2196L、F2196K、M2199A、M2199W 或 M2199R 取代的六个 B 结构域缺失（BDD）-FVIII 蛋白。与 WT-BDD-FVIII 相比，rBDD-FVIII-F2196K 和 rBDD-FVIII-M2199A 刺激的 T 细胞克隆和多克隆系的增殖减少。BDD-FVIII-F2196K 序列修饰似乎是这里测试的最有前途的序列变体，因为它能有效消除 DRB1*01:01 限制性免疫原性，在其他 MHCII 等位基因背景下具有较低的潜在免疫原性，表达水平与 WT-BDD-FVIII 相当，并且保留了促凝活性。这些结果为针对特定 HA 患者亚群设计免疫原性较低的 FVIII 蛋白提供了原理证明。
