CD40L核酸适配体诱导移植免疫耐受及分离GVHD和GVL的研究

Selectively prevention of Graft-versus-Host-Disease (GVHD) while preserving Graft-versus-Leukemia (GVL) effects is one of the major obstacles following allogeneic haematopoietic stem cell transplantation (allo-HSCT). Immune tolerance could be a promising approach to achieve this purpose. CD40L is one of the main costimulatory molecules responsible for the proper activation of donor T lymphocytes, and it has been documented that anti-CD40L antibody could effectively induced a stable mixed chimerism and facilitated donor-specific tolerance without GVHD. However, clinical application of anti-CD40L anibody has been hampered by severe thromboembolic complications. Nevertheless, aptamers have recently progressed to be the best alternative of antibodies. It may even rival monoclonal antibodies in many applications, including diagnosis and therapy. Our previous works show CD40L expression on T cells up-regulated during the occurrence of the severe GVHD, and aptamer binded to CD40L was sufficient to inhibit T cell proliferation and T cell-dependent B cell proliferation in vitro. Thus, we hypothesized that the selected CD40L aptamer for the specifically blockade of CD40L might be able to separate GVHD from the GVL effects. According to this hypothesis, our study will focus on whether CD40L expression is correlated with the development of GVHD, and the role of CD40L aptamer for immune tolerance involves the delicate balance between the desirable GVL and the undesirable GVHD. Moreover, we give an outlook on potential new diagnostic and therapeutic approaches, including the use of CD40L as clinical biomarkers and the manipulation of immune responses by CD40L aptamer.

免疫耐受是分离GVHD和GVL的理想策略。研究发现CD40L单抗可以发挥外周和中枢耐受诱导机制，建立稳定嵌合体诱导持久免疫耐受。但是，CD40L单抗的临床试验因为血栓并发症而中止，需要研发新的CD40L阻滞剂。核酸适配体的作用和抗体相似，在诊断和治疗应用中甚至优于抗体，是替代抗体的最佳选择。我们前期研究观察到allo-HSCT受者发生中重度GVHD时CD40L表达增高；初步研究显示在体外CD40L适配体可以阻断CD40与CD40L交联。基于前期工作我们提出假说：CD40L适配体可诱导持久免疫耐受，实现GVHD和GVL分离。为验证此假说，本项目拟从临床和基础二个层面开展研究，阐明CD40L及相关炎性因子与GVHD发生发展的相关性；阐明CD40L适配体诱导和维持供体T细胞的特异性免疫耐受、分离GVHD和GVL的作用。研究将为移植免疫提供新的生物标志和治疗策略，尚未见相关文献报道。

急性移植物抗宿主病（aGVHD）是异基因造血干细胞移植（allo-HSCT）术后的一种严重并发症，如何早期诊断并有效防治aGVHD是当前的重要挑战和难点问题。已有研究发现CD40-CD40L共刺激通路是启动aGVHD同种异体免疫反应的关键因素，也证实了CD40L单抗诱导免疫耐受的持久性和稳定性，但其临床试验因诱导血栓并发症而中止，迫切需要研发新的CD40L阻滞剂。核酸适配体（aptamer）的作用和抗体相似，在诊断和治疗应用中甚至优于抗体，是替代抗体的最佳选择。本研究第一部分通过分析移植相关的临床数据，发现患者并发中重度GVHD时CD40L的表达水平增高，造血重建、GVHD、肿瘤负荷、血流感染等并发症与炎性因子表达相关。第二部分以人重组CD40L蛋白为靶标，应用SELEX技术成功筛选获得具高度特异性和高度亲和力的CD40L-aptamer。初步建立了CD40L-aptamer在流式细胞术和酶联免疫吸附试验中的检测体系，灵敏度高、特异性好且简便可行。并在小鼠GVHD模型中应用活体实时荧光成像技术分析CD40L-aptamer的药代动力学特点，制定给药途径和方案。研究结果显示CD40L-aptamer可有效减轻GVHD的严重程度，改善移植生存预后，同时不影响供者嵌合状态和免疫重建。本研究为移植免疫提供早期诊断和治疗策略，并拓展了核酸适配体技术在移植免疫领域中的应用，有很好的临床意义和应用前景。