去糖基化CD36单抗用于预防CD36抗体引起的FNAIT免疫治疗研究

The study confirmed that individuals with CD36 type I deficiency may be at risk of developing anti-CD36 antibodies after receiving transfusion or during pregnancy. However, the CD36 type I deficiency in Chinese populations was about 0.5%, which is the important factor for inducing FNAIT. But the immunotherapy of FANIT caused by CD36 antibody is still unknown. This project will research for the immunotherapy of FNAIT induced by CD36 antibody, which could be prevented by deglycosylated CD36 monoclonal antibody. The murine anti-mouse CD36 mAb, IgG subtype, was obtained by platelets immunization and the preparation of monoclonal antibody. Then the glucosidase was used to hydrolyze the Fc fragment of anti-mouse CD36-IgG. Through three studies on assessment of transplacental maternofetal transport and antibody-binding characterization using SPR, platelet phagocytosis and clearance assay, to clear the antibody-binding characterization of deglycosylated anti-mouse CD36-IgG with CD36 and its placental transfer function, and to elucidate its inhibition on CD36+/+ or CD36-/+ platelet phagocytosis and clearance caused by CD36 antibody. On the basis of FNAIT mouse model induced by CD36 antibody, to demonstrate that deglycosylated anti-mouse CD36-IgG could be used to prevent the FNAIT. In totally, the performance of this project will help to supply important theoretical and technical support for clinical antenatal immunotherapy of FNAIT caused CD36 antibody.

研究证实，I型CD36缺失者经输血或怀孕过程有诱导CD36抗体产生的危险。而中国人群CD36I型缺失率约0.5%，是引起FNAIT发生的重要因素，但尚未见CD36抗体介导FNAIT发生的免疫治疗研究。本项目拟在前期研究基础上，开展去糖基化修饰CD36单抗预防CD36抗体引起的FNAIT治疗研究。通过血小板免疫和单抗制备获得IgG型抗鼠CD36单抗，以糖苷酶水解抗鼠CD36-IgG的Fc段聚糖，经抗体胎盘转运及结合特性分析，血小板吞噬和血小板清除实验这三方面研究，明确去糖基化抗鼠CD36-IgG与CD36的结合特性及其胎盘转运功能，阐明去糖基化抗鼠CD36-IgG竞争抑制CD36抗体对CD36+/+或CD36-/+血小板的吞噬清除作用，利用CD36抗体介导的FNAIT小鼠模型，证实去糖基化抗鼠CD36-IgG对FNAIT的免疫治疗效果，为今后其临床产前免疫治疗提供重要理论和技术支持。

研究证实，I型CD36缺失者经输血或怀孕过程有诱导CD36抗体产生的危险。而中国人群CD36I型缺失率约0.5%，是引起FNAIT发生的重要因素，但缺乏CD36抗体介导FNAIT发生的免疫治疗研究。本项目前期已建立CD36抗体介导的FNAIT小鼠模型，并发现母源性CD36抗体的产生可导致胎鼠流产、仔鼠出血，甚至死亡等情况。对此，本研究拟利用该模型开展去糖基化修饰CD36单抗预防CD36抗体引起的FNAIT治疗研究。本研究首先通过对CD36基因敲除（CD36-/-）小鼠免疫及单抗制备技术，获得抗鼠CD36单克隆抗体（32-106），并将32-106单抗经尾静脉注射给已孕CD36-/-小鼠，建立被动的FNAIT小鼠模型，进一步证实CD36抗体介导FNAIT发生的作用机制。其次，本研究将分离纯化的32-106单抗进行去糖基化修饰，通过体内实验表明32-106单抗及去糖基化32-106的胎盘转运及血小板清除机制；体外实验证实去糖基化32-106与母源性CD36抗体的竞争抑制结合血小板CD36的作用机制，为进一步产前免疫治疗研究奠定基础。最后，将去糖基化32-106单抗用于CD36抗体引起的FNAIT小鼠模型治疗研究，发现去糖基化CD36单抗可明显减少胎鼠死亡率及提高仔鼠的血小板数量。然而，晚期IVIG治疗（妊娠10d，15d，20d）并不能缓解胎鼠的死亡率。本研究证实了去糖基化CD36单抗对CD36抗体引起的FNAIT的免疫治疗潜能，为今后其临床免疫治疗研究提供重要理论和技术支持。

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