凝血因子IX Ser411Gly突变导致血友病B患者抑制物形成的分子机制

Haemophilia B is a hereditary hemorrhagic disease caused by coagulation factor Ⅸ (FⅨ) deficiency. Exogenous FⅨ concentrates replacement therapy is currently the mainstream of treatment. However, the development of coagulation factor-related inhibitors is the main problem of haemophilia B treatment. Identification of the molecular mechanism of FⅨ inhibitor development is a prerequisite for timely diagnosis and for the development of FⅨ concentrates for individualized treatment. We have previously found that FⅨ Ser411Gly mutation led to CRM+ severe haemophilia B, and the patient developed high titer of FⅨ inhibitor with IgG4 isoform after infusion of exogenous FⅨ concentrates, presuming that FⅨ 411 amino acid site plays an important role in inhibitor development, and possibly is an important recognition epitope of T / B cell in the process of inhibitor formation. In the current project, on the basis of successfully constructing humanized FⅨ wild-type and mutant (Ser411Gly) mice models，as well as obtaining recombinant mutant FⅨ protein through amino acid alteration at FⅨ 411 amino acid site, we aim to clearify the immunogenicity of inhibitor development caused by FⅨ Ser411Gly mutation and identify whether FⅨ Ser411 is an important recognition epitope of the T / B cell during the process of FⅨ inhibitor develoment. Meanwhile, the influence of mutation at FⅨ 411 amino acid site on the immunogenicity of FⅨ protein will also be explored. The clearification of above issues will provide a theoretical basis and target for the clinical diagnosis and research and development (R&D) of low immunogenic FⅨ concentrates with independent intellectual property rights.

血友病B是凝血因子Ⅸ（FⅨ）缺陷导致的遗传性出血性疾病，外源性FⅨ制剂替代治疗是主要治疗方式，但凝血因子相关抑制物的产生是困扰血友病B治疗最主要问题。明确血友病B患者FⅨ抑制物产生的分子机制是及时诊断和研发个体化治疗用FⅨ制剂的前提。申请人前期发现FⅨSer411Gly突变可导致CRM+重型血友病B，患者在输注外源性FⅨ制剂后产生高滴度IgG4型FⅨ抑制物，推测FⅨ411氨基酸位点在抑制物产生中扮演重要角色，可能是抑制物形成中T/B细胞重要识别表位。本项目拟在成功构建人源化FⅨ野生型和突变型（Ser411Gly）小鼠模型及获得该位点突变氨基酸重组蛋白基础上，明确FⅨSer411Gly突变导致抑制物产生的免疫特性及该氨基酸位点在T/B细胞识别中的作用，同时探讨FⅨ411氨基酸位点突变对FⅨ蛋白免疫原性影响。以上问题的明确将为临床及时诊断和研发具有自主知识产权低免疫原性FⅨ制剂提供依据和靶点。

血友病B是凝血因子Ⅸ（FⅨ）缺陷导致的遗传性出血性疾病，外源性FⅨ制剂替代治疗是主要治疗方式，但凝血因子相关抑制物的产生是困扰血友病B治疗最主要问题。明确血友病B患者FⅨ抑制物产生的分子机制是及时诊断和研发个体化治疗用FⅨ制剂的前提。申请人前期发现 FⅨSer411Gly突变可导致CRM+重型血友病B，患者在输注外源性FⅨ制剂后产生高滴度IgG4型FⅨ抑制物，推测FⅨ411氨基酸位点在抑制物产生中扮演重要角色，可能是抑制物形成中T/B细胞重要识别表位。本项目成功构建人源化FⅨ野生型和突变型（Ser411Gly）小鼠模型，并对其进行了人重组FIX制剂（BeneFIX）免疫，发现约30%的人源化突变小鼠可产生低滴度IgG1型和IgG2a型FIX抑制物，相当于患者产生的IgG4型FIX抑制物。抑制物阳性小鼠的Treg水平和FoxP3 mRNA水平均显著低于抑制物阴性小鼠，提示FIX-S411G小鼠输注外源性BeneFIX获得抑制物的过程是由于机体免疫抑制能力降低所致。抑制物阳性小鼠体内T-bet，GATA-3和RoRrt三个基因的mRNA转录水平均显著升高，提示FIX抑制物的生成是Th细胞所介导的。与此同时，迅速增高的IL-2和IL-10提示其在抑制物形成中发挥着重要的作用。我们对经BeneFIX皮下免疫的FIX-S411G小鼠的淋巴结T细胞进行体外再刺激研究，发现经BeneFIX和WT-1短肽再刺激后T细胞增殖率均会显著提高，并以CD4+T细胞增殖为主，进一步证实FIX抑制物的生成是Th细胞所介导的，且含Ser411位点的WT-1肽段是抑制物形成过程中T细胞的识别表位所在。我们对经BeneFIX静脉免疫的FIX-S411G小鼠细胞进行记忆B细胞体外再刺激研究，发现经BeneFIX和WT-2短肽再刺激后更多的B细胞可分泌抗体，证实含Ser411位点的WT-2肽段是抑制物形成过程中B细胞的识别表位所在。综上所述，本研究明确FⅨSer411Gly突变导致抑制物产生的免疫特性及该氨基酸位点在T/B细胞识别中的作用，为临床及时诊断和研发具有自主知识产权低免疫原性FⅨ制剂提供依据和靶点。