Department of Pediatrics and Adolescent Medicine, University Hospital, Goethe University, Frankfurt am Main, Germany.
Faculty of Biological Science, Goethe University, Frankfurt am Main, Germany.
J Thromb Haemost. 2018 Nov;16(11):2223-2232. doi: 10.1111/jth.14273. Epub 2018 Oct 8.
Essentials There is still a need for novel therapeutic approaches for hemophilia A patients with inhibitors. A factor VIII domain was used as the targeting moiety for elimination of FVIII-specific B cells. The immunodominant C2 domain was fused to exotoxin A from Pseudomonas aeruginosa (hC2-ETA). Murine C2 domain-specific B cells were selectively and efficiently eliminated by hC2-ETA ex vivo. SUMMARY: Background Today, the most serious complication for patients with hemophilia A undergoing factor VIII (FVIII) replacement therapy is the development of neutralizing antibodies (inhibitors). Although inhibitors can be eradicated by application of high doses of FVIII, the immune tolerance induction therapy fails in up to 30% of patients. Hence, there is still an urgent need for novel therapeutic approaches for patients with persisting inhibitors. Objectives In the present study, the potential use of immunotoxins containing exotoxin A (ETA) from Pseudomonas aeruginosa for selective elimination of FVIII-specific B cells was explored. Methods The immunodominant C2 domain of human FVIII was used as a targeting moiety instead of the full-length FVIII protein and the resulting human C2 domain-ETA fusion protein (hC2-ETA) was produced in Escherichia coli. Results Binding studies with monoclonal C2 domain-specific antibodies confirmed the conformational integrity of the C2 domain in hC2-ETA. The functionality of hC2-ETA was tested ex vivo by incubation of splenocytes from inhibitor-positive FVIII knockout mice with hC2-ETA and controls. FVIII-specific memory B cells from splenocytes were differentiated by FVIII stimulation in antibody-secreting cells (ASC) and detected by an enzyme-linked immunospot assay. Although the controls showed no effect, incubation of splenocytes with hC2-ETA reduced the number of C2-specific ASC in a dose-dependent fashion, indicating specific and efficient elimination of C2-specific memory B cells. Conclusions Overall, the results of the study support the fact that FVIII domain immunotoxins might be a potential new tool for the elimination of FVIII-specific B cells in patients with hemophilia A and persisting inhibitors.
今天，接受凝血因子 VIII（FVIII）替代治疗的血友病 A 患者最严重的并发症是产生中和抗体（抑制剂）。尽管通过应用高剂量的 FVIII 可以消除抑制剂，但免疫耐受诱导疗法在多达 30%的患者中失败。因此，对于持续存在抑制剂的患者，仍然迫切需要新的治疗方法。
本研究探讨了含有铜绿假单胞菌外毒素 A（ETA）的免疫毒素用于选择性消除 FVIII 特异性 B 细胞的潜在用途。
用人 FVIII 的免疫显性 C2 结构域代替全长 FVIII 蛋白作为靶向部分，并在大肠杆菌中生产出人 C2 结构域-ETA 融合蛋白（hC2-ETA）。
用单克隆 C2 结构域特异性抗体进行的结合研究证实了 hC2-ETA 中 C2 结构域的构象完整性。通过将抑制剂阳性 FVIII 敲除小鼠的脾细胞与 hC2-ETA 和对照物孵育，在体外测试 hC2-ETA 的功能。通过 FVIII 刺激将脾细胞中的 FVIII 特异性记忆 B 细胞分化为抗体分泌细胞（ASC），并通过酶联免疫斑点测定法检测。尽管对照物没有效果，但 hC2-ETA 孵育以剂量依赖性方式减少了 C2 特异性 ASC 的数量，表明 C2 特异性记忆 B 细胞的特异性和有效消除。
总体而言，研究结果支持 FVIII 结构域免疫毒素可能是消除血友病 A 患者和持续存在抑制剂的 FVIII 特异性 B 细胞的潜在新工具的事实。
