a Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology , China Pharmaceutical University , Nanjing , China.
MAbs. 2019 Feb/Mar;11(2):401-410. doi: 10.1080/19420862.2018.1537580. Epub 2018 Dec 22.
In vitro immunization 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 (pNOPhe), which stimulated an in vitro immune response in human immune cells. After constructing an antigen-specific antibody library from in vitro immunized 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 maturation in 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 with pNOPhe.
体外免疫可用于产生单克隆抗体（mAbs），但该技术受到较差的重现性和预免疫淋巴细胞的要求的限制。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产生针对肿瘤坏死因子（TNF）的人 IgG 中的应用。我们表达了具有定点掺入对硝基苯丙氨酸（pNOPhe）的突变蛋白，该蛋白刺激了人类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文库后，我们证明了在体外亲和力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。为了模拟类别转换，我们通过噬菌体展示方法筛选高亲和力的抗原结合片段，对它们进行组装并鉴定出中和人 TNF 的人 IgG。这种方法可能为针对自身蛋白产生高亲和力单克隆抗体提供一种通用方法。此外，它支持使用 pNOPhe 打破人类自身耐受的机制理解。
