Monoclonal antibody (mAb) drugs are clinically important for the treatment of various diseases. TTAC-0001 is under development as a new anti-cancer antibody drug targeting VEGFR-2. As the less severe toxicity of TTAC-0001 compared to Bevacizumab, likely due to the decreased in vivo half-life, seems to be related to its structural flexibility, it is important to map the exact flexible regions. Although the 13C/15N-labeled protein is required for NMR analyses, it is difficult to obtain antibody fragments (Fab and scFv) containing disulfide bonds through general cytosolic expression in Escherichia coli (E. coli). Here, we notably increased the periplasmic expression of the 13C/15N-labeled TTAC-0001-Fab (13C/15N-TTAC-Fab) through simple isopropyl β-D-1-thiogalactopyranoside (IPTG)-induction at an increased optical density (1.5 OD600nm). Through NMR triple resonance experiments, two loop insertions (LI-1 between the VH and CH1; LI-2 between the VL and CL) were confirmed to be highly flexible. The additional LIs could be another way to engineer the antibody by changing the pharmacokinetic properties.

中文翻译：

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13C / 15N标记的抗体TTAC-0001-Fab的高度柔性区域的NMR绘图。

单克隆抗体（mAb）药物在治疗各种疾病方面具有重要的临床意义。TTAC-0001作为针对VEGFR-2的新型抗癌抗体药物正在开发中。由于与贝伐珠单抗相比，TTAC-0001的毒性较轻（可能由于体内半衰期缩短）似乎与其结构柔性有关，因此绘制精确的柔性区域非常重要。尽管13C / 15N标记的蛋白是NMR分析所必需的，但是很难通过在大肠杆菌（E. coli）中通过一般的胞质表达来获得包含二硫键的抗体片段（Fab和scFv）。在这里，我们通过简单的异丙基β-D-1-硫代吡喃半乳糖吡喃糖苷（IPTG）诱导以增加的光密度显着增加了13C / 15N标记的TTAC-0001-Fab（13C / 15N-TTAC-Fab）的周质表达（ 1.5 OD600nm）。通过NMR三重共振实验，证实两个环插入（VH和CH1之间的LI-1； VL和CL之间的LI-2）具有高度的柔性。额外的LIs可能是通过改变药代动力学特性来改造抗体的另一种方法。