Abstract

Due to their potent immune stimulation, tumor necrosis factor alpha (TNFα) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNFα in clinical trials triggered extensive interest in developing novel tumor-homing TNFα variants in recent years. Owing to its promising antitumor effect, NGR-TNFα is usually used as a control for newly developed tumor-homing TNFα variants. In our previous works, we produced a pericyte-targeting Z-TNFα at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNFα and NGR-TNFα, we attempted to express NGR-TNFα using the same system. Surprisingly, native NGR-TNFα was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNFα, increased the expression of NGR-TNFα by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNFα. As fusing NGR to the N-terminus of TNFα with a valine substitution did not reduce the protein yield, the TNFα gene with a C > G mutation might be used to prepare novel tumor-homing TNFα when the native TNFα-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNFα variant must be carefully evaluated.

Key points

• A single nucleotide mutation increased the expression of NGR-TNFα by two orders.

• Nucleotide mutation-induced amino acid substitution did not reduce NGR-TNFα activity.

中文翻译：

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通过改善基因转录，单核苷酸突变可大大增加大肠杆菌M15-pQE30系统中归巢的NGR-TNFα的表达

摘要

由于其有效的免疫刺激作用，具有肿瘤归巢活性的肿瘤坏死因子α（TNFα）变体作为新型抗肿瘤药物具有吸引力。NGR-TNFα在临床试验中有希望的抗肿瘤作用引起了近年来对开发新型肿瘤归巢TNFα变体的广泛兴趣。由于其有希望的抗肿瘤作用，NGR-TNFα通常用作新开发的肿瘤归巢TNFα变体的对照。在我们以前的工作中，我们使用大肠杆菌（E. coli）M15-pQE30系统高水平生产了靶向周细胞的Z-TNFα 。为了进一步比较Z-TNFα和NGR-TNFα，我们尝试使用同一系统表达NGR-TNFα。令人惊讶的是，天然NGR-TNFα在大肠杆菌中以低水平（〜0.2 mg / L）表达含有pQE30质粒的M15。但是，C到G的单核苷酸突变导致TNFα起始处亮氨酸（L）被缬氨酸（V）取代，通过改善转录，NGR-TNFα的表达增加了约100倍。另外，氨基酸取代对NGR-TNFα的受体结合，体外细胞毒性和体内抗肿瘤作用几乎没有影响。由于将NGR融合到具有缬氨酸取代的TNFα的N端不会降低蛋白质产量，因此当天然TNFα基变体表达于C时，具有C> G突变的TNFα基因可用于制备新型的肿瘤归巢TNFα。大肠杆菌中的极低水平。 值得注意的是，除了突变的缬氨酸外，还必须仔细评估pQE30载体提供的N末端其他氨基酸对TNFα变体功能的影响。

关键点

•单核苷酸突变使NGR-TNFα的表达增加了两个数量级。

•核苷酸突变诱导的氨基酸取代不会降低NGR-TNFα活性。