De novo generation of specific human IgGs by in vitro immunization using autologous proteins containing immunogenic p-nitrophenylalanine
De novo generation of specific human IgGs by in vitro immunization using autologous proteins containing immunogenic p-nitrophenylalanine
复制标题
使用含有免疫原性对硝基苯丙氨酸的自体蛋白通过体外免疫从头生成特定的人 IgG
DOI：
10.1080/19420862.2018.1537580
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发表时间：
2019
期刊：
影响因子：
5.3
通讯作者：
Yao Wenbing
中科院分区：
文献类型：
--
作者：
Tong Yue;Fang Xu;Tian Hong;Zhong Shengwei;Jin Liang;Gao Xiangdong;Yao Wenbing
体外免疫技术可用于制备单抗，但由于重复性差和预先免疫淋巴细胞的要求而受到限制。为了改进这种方法，我们最近开发了一种快速产生抗原特异性B细胞的方法。在这里，我们报告了该系统在生产抗肿瘤坏死因子(TNF)的人免疫球蛋白方面的应用。我们用位点特异性掺入的p-硝基苯丙氨酸(PNO2Phe)表达了突变蛋白，它能刺激人免疫细胞的体外免疫反应。在用荧光激活细胞分选鉴定的体外免疫B细胞构建抗原特异性抗体库后，我们证明了在我们的体外亲和成熟的逐步共培养系统中发生了许多可变区的点突变事件。为了模拟类的转换，我们通过噬菌体展示的方法寻找高亲和力的抗原结合片段，将它们组装在一起，鉴定出hTNF中和人免疫球蛋白。该方法为制备高亲和力的抗自身蛋白单抗提供了一种通用的方法。此外，它还支持对用pNO2Phe打破人类自我耐受的机械论理解。
In vitroimmunization can to used to produce monoclonal antibodies(mAbs), but this technology is limited by poor reproducibility and the requirement of pre-immunized lymphocytes. To improve this approach, we recently developed a method for rapid generation of antigen-specific B cells. Here, we report the application of this system to the production of human IgGs against tumor necrosis factor (TNF). We expressed mutant proteins with site-specific incorporated p-nitrophenylalanine (pNO2Phe), which stimulated anin vitroimmune response in human immune cells. After constructing an antigen-specific antibody library fromin vitroimmunized B cells identified by fluorescence-activated cell sorting, we demonstrated that many point mutation events of the variable region occurred in our step-by-step co-cultivation system for affinity maturationin vitro. To mimic the class switching, we panned for high-affinity antigen-binding fragments by the phage display method, assembled them and identified hTNF-neutralizing human IgGs. This approach may provide a general method for raising high-affinity monoclonal antibodies against self-proteins. Furthermore, it supports mechanistic understanding in breaking human self-tolerance withpNO2Phe.