抗TIGIT单克隆抗体的制备及活性鉴定

目的制备新型抑制剂膜分子TIGIT(T cell immunoglobulin and ITIM domain)的单克隆抗体并鉴定其在心脏移植免疫耐受中的作用.方法用人TIGIT-GST重组蛋白免疫小鼠并制备单克隆抗体,抗体特异性实验检测抗体与TIGIT的结合能力.运用重导向杀伤实验检测TIGIT抗体对NK细胞杀伤P815的作用.通过酶吸收法检测TIGIT活化性抗体对CTLs及CD8 Treg细胞活性的影响.结果成功制备得到一株非活化的抗TIGIT的抗体7M13和一株活化性的抗TIGIT抗体4D9.抗体特异性实验表明抗体7M13和4D9均能与人和小鼠的TIGIT结合.重导向杀伤实验表明该抗体7M13对NK细胞杀伤P815细胞的功能没有显著抑制作用,说明7M13可以与TIGIT结合而不活化TIGIT;而4D9则可以显著抑制NK细胞的杀伤功能.从37例心脏移植患者中分离CTLs及CD8 Treg,采用TIGIT活化性抗体处理对细胞生存能力无显著影响,但可明显抑制CTLs的增殖能力,而对CD8 Treg的增殖能力无显著影响.结论分别成功获得非活化的抗TIGIT抗体7M13和活化性的抗TIGIT抗体4D9,并且4D9可显著抑制NK细胞的杀伤功能.

Preparation and Activity Identification of Anti-TIGIT Monoclonal An-tibody

Objective A monoclonal antibody against TIGIT was prepared and its effects on cardiac allograft tolerance was examined.Methods Mice were immunized with human TIGIT-GST recombinant protein and monoclonal antibodies were prepared.Antibody specificity assay was used to detect the binding ability of antibodies against TIGIT.The effect of TIGIT antibody on the killing of P815 by NK cells was examined by using a redirected killing assay.The effect of TIGIT ac-tivating antibodies on the activity of CTLs and CD8 Treg cells was examined by enzyme absorption assay.Results A non-activated anti-TIGIT antibody 7M13 and an activated anti-TIGIT antibody 4D9 were successfully prepared.Antibody specificity experiments showed that both antibodies 7M13 and 4D9 bound to TIGIT in humans and mice.The retargeting killing experiment showed that the an-tibody 7M13 did not significantly inhibit the killing effect of NK cells on P815 cells,indicating that 7M13 can bind to TIGIT without activating TIGIT while 4D9 can significantly inhibit the killing func-tion of NK cells.The isolation of CTLs and CD8 Treg from 37 heart transplant patients,TIGIT acti-vating antibody treatment had no significant effects on cell viability,but significantly inhibited the proliferation of CTLs,but exerted no significant effect on the proliferation of CD8 Treg cells.Conclusion The non-activated anti-TIGIT antibody 7M13 and the activated anti-TIGIT anti-body 4D9 were successfully prepared,respectively,and 4D9 could significantly inhibit the killing function of NK cells.