Akbarzadeh-Sharbaf Soudabeh, Yakhchali Bagher, Minuchehr Zarrin, Shokrgozar Mohammad Ali, Zeinali Sirous
Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology, (NIGEB), Tehran, Iran.
Avicenna J Med Biotechnol. 2013 Apr;5(2):87-95.
Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb.
According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method.
Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l /day. According to the results, the produced mAb had similar affinity to HER2(+) tumor cells to that of Herceptin.
High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase (DHFR). It is usually accepted that DHFR gene can be amplified in DHFR(－) CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR.
曲妥珠单抗（赫赛汀）是一种人源化单克隆抗体（mAb），用于特异性治疗HER2/neu受体过表达的转移性乳腺癌患者。在本研究中，我们试图开发一种曲妥珠单抗mAb的生物类似药。
根据计算机模拟研究，设计并构建了曲妥珠单抗mAb的重链和轻链。将重组构建体共转染到CHO DG44细胞系中。在半固体培养基上选择稳定的转化体。通过甲氨蝶呤实现重链基因扩增的基因组扩增。通过流式细胞术方法测试所产生抗体与赫赛汀相比的生物活性。
经过七轮甲氨蝶呤处理后获得了三倍的扩增。结果表明重链和轻链具有相等的表达水平。纯化的mAb产量为每天50至60mg/l。根据结果，所产生的mAb与HER2（+）肿瘤细胞的亲和力与赫赛汀相似。
通过用可选择标记物如二氢叶酸还原酶（DHFR）扩增重组基因，可以实现高水平的重组蛋白表达。通常认为DHFR基因可以在DHFR（-）CHO细胞中扩增，从而导致共连接的靶基因扩增，最终实现重组蛋白的扩增。在本研究中，为了生产赫赛汀的生物类似药，使用定量实时PCR研究了基因组扩增对增加基因拷贝数的影响。
