Shariaty Vaziri Zahra, Shafiee Fatemeh, Akbari Vajihe
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
Biotechnol Lett. 2023 Apr;45(4):537-550. doi: 10.1007/s10529-023-03360-4. Epub 2023 Feb 20.
Human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of some kinds of cancer cells including breast cancer. In this study, we designed and produced a novel immunotoxin consisting anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified form of Pseudomonas exotoxin (PE35KDEL).
The three-dimensional (3D) structure of the fusion protein (anti-HER IT) was predicted by MODELLER 9.23 and its interaction with HER2 receptor was assessed using HADDOCK web server. Anti-HER2 IT, anti-HER2 scFv, and PE35KDEL proteins were expressed by Escherichia coli BL21 (DE3). After purification of the proteins using Ni affinity chromatography and refolding through dialysis, the cytotoxicity of proteins against breast cancer cell lines was examined by MTT assay.
In-silico studies showed that (EAAAK)2 linker can efficiently prevent the formation of salt bridges between two functional domains and the constructed fusion protein has a high affinity to HER2 receptor. The optimum condition of anti-HER2 IT expression was 25 °C and 1 mM IPTG. The protein was successfully purified and refolded by dialysis with a final yield of 45.7 mg per 1 L of bacterial culture. The cytotoxicity results showed that anti-HER2 IT was much more toxic on HER2-overexpressing cells, BT-474 (IC ~ 95 nM) compared with HER2-negative cells, MDA-MB-23 (IC ˃ 200 nM).
This novel immunotoxin has the potential to be applied as a therapeutic candidate for HER2-targeted cancer therapy. However further in vitro and in vivo evaluations are still required to confirm the efficacy and safety of this protein.
人表皮生长因子受体2（HER2）在包括乳腺癌在内的某些癌细胞表面过度表达。在本研究中，我们设计并制备了一种新型免疫毒素，其由帕妥珠单抗的抗HER2单链Fv（scFv）和修饰形式的铜绿假单胞菌外毒素（PE35KDEL）组成。
利用MODELLER 9.23预测融合蛋白（抗HER免疫毒素）的三维（3D）结构，并使用HADDOCK网络服务器评估其与HER2受体的相互作用。抗HER2免疫毒素、抗HER2 scFv和PE35KDEL蛋白由大肠杆菌BL21（DE3）表达。使用镍亲和色谱法纯化蛋白质并通过透析进行复性后，通过MTT法检测蛋白质对乳腺癌细胞系的细胞毒性。
计算机模拟研究表明，（EAAAK）2接头可以有效防止两个功能域之间形成盐桥，并且构建的融合蛋白对HER2受体具有高亲和力。抗HER2免疫毒素表达的最佳条件是25℃和1 mM IPTG。该蛋白通过透析成功纯化并复性，每1 L细菌培养物的最终产量为45.7 mg。细胞毒性结果表明，与HER2阴性细胞MDA-MB-23（IC＞200 nM）相比，抗HER2免疫毒素对HER2过表达细胞BT-474（IC～95 nM）的毒性要大得多。
这种新型免疫毒素有潜力作为HER2靶向癌症治疗的候选治疗药物。然而，仍需要进一步的体外和体内评估来确认该蛋白的疗效和安全性。
