Agha Amiri Solmaz, Shahhosseini Soraya, Zarei Najmeh, Khorasanizadeh Dorsa, Aminollahi Elahe, Rezaie Faegheh, Zargari Mehryar, Azizi Mohammad, Khalaj Vahid
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
AMB Express. 2017 Dec;7(1):112. doi: 10.1186/s13568-017-0410-5. Epub 2017 Jun 2.
CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.
CD22标记物是一种高度内化的抗原，位于B细胞表面，正被用作治疗B细胞恶性肿瘤的一个有前景的靶点。靶向CD22的单克隆抗体已被引入，其中一些目前正在临床试验中进行研究。基于抗体药物偶联物的成功，我们开发了一种由新型抗CD22单链抗体片段（scFv）和凋亡素组成的融合蛋白，并在淋巴瘤细胞中测试了其结合和治疗效果。该重组蛋白在大肠杆菌中表达，并成功纯化和复性。通过免疫荧光和流式细胞术进行的体外结合分析表明，该重组蛋白特异性结合CD22阳性的Raji细胞，而不结合CD22阴性的Jurkat细胞。通过MTT法和膜联蛋白V/碘化丙啶流式细胞术分析评估了scFv-凋亡素的细胞毒性特性，结果表明该重组蛋白优先诱导Raji细胞凋亡，而在Jurkat细胞中未检测到明显作用。我们的研究结果表明，重组抗CD22 scFv-凋亡素融合蛋白能够成功穿过细胞膜并以高特异性诱导凋亡，使其成为B细胞恶性肿瘤免疫治疗的一个有前景的分子。
