BCR靶向体介导allo-B淋巴细胞克隆清除的体液免疫耐受研究

B lymphocyte-mediated humoral rejection affects the long-term survival of transplanted organs and an immune tolerance regimen for transplant rejection was not established yet. Organ transplantation is the most effective way to treat end-stage organ failure, but high risk of post-transplant rejection is still a major challenge in clinic. The anti-donor-specific antibodies (DSA) that mediate humoral rejection are produced by allo-B lymphocytes, whose idiotype of B-cell receptor (allo-BCR) is the surface-specific marker. We aim to achieve the humoral immune tolerance by designing a construct which consisting of allo-BCR idiotype specific targeting peptide and human IgG-Fc domain to eliminate the allo-reactive B cell clones specifically. To screen the peptides for the BCR idiotype binding , we initially establish the BCR-like library by the isolated and purified allo-antibodies (IgG) from the recipients with organ transplant rejection. High-throughput screening of allo-BCR-targeted polypeptides are performed and verified by the imitated BCR array and the real BCR-expressing cells. Then, we make BCR-targeting peptide-bodies and test its effect on the elimination of allo-reactive B cells in vitro and in xeno-tansplant SCID mice model, as well as understand the mechanism of its establishment of the humoral immune tolerance. The success of this project will create a novel therapy to reduce the post-transplant rejection and prolong the survival of the allograft, which has a great clinical significance.

同种异型反应性B淋巴细胞（allo-B）是介导移植排斥的重要因素，当前临床移植尚缺乏有效的免疫耐受方法。引起抗体介导排斥反应（AMR）的抗供体特异抗体(DSA)是由allo-B产生的，而产生DSA的B细胞的BCR独特型是该allo-B细胞克隆的表面标志。为了筛选与allo-BCR靶向结合的多肽分子，取移植病例的allo-抗体IgG制备模拟allo-BCR文库，再对allo-抗原的表位多肽进行高通量筛选。对筛选出的靶肽与人IgG-Fc片段融合制成BCR靶向体。1)测试其对allo-B细胞模型的体外特异性靶向杀伤效应；2) 测试其对SCID小鼠模型体内转移的allo-B细胞克隆清除能力；3) 探究BCR靶向体对allo-B细胞克隆清除的免疫耐受机制。为建立器官移植的体液免疫耐受开创新思路和突破口。本项目研究的成功将带来临床抗移植排斥和延长移植物存活时间的全新治疗方案，具有重要的临床医学意义。

本项研究聚焦于引起抗体介导排斥反应（AMR）的特异性allo-B淋巴细胞，将产生抗供者特异性抗体（DSA）的B淋巴细胞的BCR独特型表面分子作为靶向清除的靶点；设计制备了3种针对allo-BCR的靶向体重组蛋白质分子；在测试杀伤效果时进行了优化，形成了可通用的可置换成不同靶向肽的靶向肽的新工艺，建立了以特异性抗体IgG标记Luminex微球获得模拟BCR用于对靶向体的靶向多肽的筛选与优化，发明专利在申请中。另外为验证靶向体的特异性清除特定B细胞的需要，确定了3种BCR细胞模型：①产生单克隆抗体的杂交瘤细胞；②EBV永生化的人源性B淋巴细胞；③人源化小鼠B淋巴细胞；为开展allo-B细胞免疫耐受研究奠定了坚实的基础。本课题研究获得了应用靶向体清除特异性B淋巴细胞的效果，为进一步推进临床研究提供了科学依据；完成本项目研究，我们发表了4篇SCI论文，获得国家发明专利(接受)1项，培养了3名博士研究生和2名硕士研究生毕业。