Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Mol Immunol. 2014 May;59(1):100-9. doi: 10.1016/j.molimm.2014.01.007. Epub 2014 Feb 14.
Targeted therapy is an attractive approach to avoid the side effects of cancer treatment. Based on antibody-targeted superantigens, single chain variable fragment (scFv) and single domain (sdAb) antibodies, characterized by a low molecular weight, low immunogenicity and a high tumor penetration compared to monoclonal antibodies (mAb), have been increasingly used in gene-targeted therapy for cancer. In the present study, we aimed to develop the novel recombinant scFv-RP215 and sdAb-RP215 antibodies based on the variable regions of the RP215 monoclonal antibody (RP215-mAb) against CA215, a pan cancer marker expressed in various human tumor tissues, and to examine their biological activity in breast cancer cell lines.
The VH and VL genes were amplified from hybridoma cells secreting RP215-mAb by RT-PCR and joined with a linker using splicing by overlap extension PCR (SOE-PCR) to obtain the RP215-scFv gene, whereas the VH gene was used to generate the RP215-sdAb. Gene fragments of antibodies were subcloned into the pET32a(+) vector and expressed in Escherichia coli BL21. Western blot, indirect immunofluorescence (IF), ELISA and competitive ELISA were used to detect the immunoreactivity of scFv-RP215, sdAb-RP215, and RP215-mAb. The CCK-8 assay and cell cycle analysis were used to assess antibodies function.
The novel recombinant scFv-RP215 and sdAb-RP215 antibodies were successfully developed based on the variable regions of the monoclonal antibody RP215 (RP215-mAb) against CA215. We verified that scFv-RP215 and sdAb-RP215 recognize CA215 on the surface of breast cancer cells (MB231, MCF7, MB468, SK-BR-3 and BT549) and characterized their activity and specificity. Our findings also indicate that scFv-RP215 and sdAb-RP215 induce cell cycle arrest at the G0/G1 phase in breast cancer cells.
Our results showed that scFv-RP215 and sdAb-RP215 have excellent immunoreactivity and localize accurately to breast cancer cells in membrane-bound form, suggesting their potential as tumor targeting antibodies for breast cancer therapy.
靶向治疗是一种避免癌症治疗副作用的有吸引力的方法。与单克隆抗体（mAb）相比，基于抗体靶向超抗原、单链可变片段（scFv）和单域（sdAb）抗体的小分子、低免疫原性和高肿瘤穿透性，已越来越多地用于癌症的基因靶向治疗。在本研究中，我们旨在基于针对 CA215 的 RP215 单克隆抗体（RP215-mAb）的可变区，开发新型重组 scFv-RP215 和 sdAb-RP215 抗体，CA215 是一种在各种人类肿瘤组织中表达的泛癌标志物，并研究它们在乳腺癌细胞系中的生物学活性。
通过 RT-PCR 从分泌 RP215-mAb 的杂交瘤细胞中扩增 VH 和 VL 基因，然后通过拼接重叠延伸 PCR（SOE-PCR）将它们与接头连接，以获得 RP215-scFv 基因，而 VH 基因则用于生成 RP215-sdAb。抗体的基因片段被亚克隆到 pET32a(+)载体中，并在大肠杆菌 BL21 中表达。使用 Western blot、间接免疫荧光（IF）、ELISA 和竞争 ELISA 检测 scFv-RP215、sdAb-RP215 和 RP215-mAb 的免疫反应性。使用 CCK-8 测定法和细胞周期分析来评估抗体的功能。
我们成功地基于针对 CA215 的单克隆抗体 RP215（RP215-mAb）的可变区，开发了新型重组 scFv-RP215 和 sdAb-RP215 抗体。我们验证了 scFv-RP215 和 sdAb-RP215 可以识别乳腺癌细胞（MB231、MCF7、MB468、SK-BR-3 和 BT549）表面的 CA215，并对其活性和特异性进行了表征。我们的研究结果还表明，scFv-RP215 和 sdAb-RP215 诱导乳腺癌细胞停滞在 G0/G1 期。
我们的结果表明，scFv-RP215 和 sdAb-RP215 具有优异的免疫反应性，并且以膜结合形式准确定位于乳腺癌细胞，这表明它们有作为乳腺癌治疗的肿瘤靶向抗体的潜力。
