Schmidt Anja, Brettschneider Kerstin, Kahle Jörg, Orlowski Aleksander, Becker-Peters Karin, Stichel Diana, Schulze Jörg, Braner Markus, Tampé Robert, Schwabe Dirk, Königs Christoph
Christoph Königs, Goethe University, Department of Paediatrics, Molecular Haemostasis and Immunodeficiency, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany, Tel.: +49 69 6301 83030, Fax: +49 69 6301 83991, E-mail:
Thromb Haemost. 2016 Jul 4;116(1):32-41. doi: 10.1160/TH15-12-0925. Epub 2016 Mar 24.
Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.
在用凝血因子VIII（FVIII）进行替代治疗后，高达30%的甲型血友病患者会产生FVIII特异性抑制性抗体（FVIII抑制剂）。免疫耐受诱导（ITI）并非总是成功，这使得FVIII抑制剂阳性患者需要替代治疗。由于ITI过程中的耐受诱导似乎涉及针对抗FVIII抗体的抗独特型的形成，这种抗独特型可能用于恢复患有FVIII抑制剂的甲型血友病患者的止血功能。我们从噬菌体展示文库中分离出与鼠源FVIII抑制剂2-76和2-77结合的抗独特型抗体片段（单链抗体）。FVIII抑制剂/抗独特型相互作用非常特异，未观察到与其他FVIII抑制剂或同型对照的交叉反应。单链抗体在体外和单克隆小鼠模型中阻断了FVIII抑制剂与FVIII的结合并中和了其同源抑制剂。此外，对FVIII抑制剂2-76特异的单链抗体JkH5对产生2-76的杂交瘤细胞进行了染色。使用JkH5丙氨酸突变体确定位于互补决定区的JkH5残基R52和Y226对于JkH5/2-76相互作用至关重要。表面等离子体共振光谱显示JkH5以纳摩尔亲和力与FVIII抑制剂2-76相互作用。因此，可以从噬菌体展示文库中分离出FVIII抑制剂特异性、高亲和力的抗独特型，并中和其各自的抑制剂。此外，我们表明抗独特型单链抗体可用于特异性靶向抑制剂特异性B细胞。因此，一组抗独特型能够在患者存在FVIII抑制剂的情况下重新建立止血功能，甚至通过靶向抑制剂特异性B细胞群体来消耗抑制剂。
