Bagheri Salman, Yousefi Mehdi, Safaie Qamsari Elmira, Riazi-Rad Farhad, Abolhassani Mohsen, Younesi Vahid, Dorostkar Ruhollah, Movassaghpour Ali Akbar, Sharifzadeh Zahra
1 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
2 Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
Tumour Biol. 2017 Mar;39(3):1010428317695924. doi: 10.1177/1010428317695924.
The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.
4-1BB是一种属于肿瘤坏死因子受体家族的表面糖蛋白。有确凿证据表明4-1BB在免疫反应中发挥重要作用，包括细胞活化、增殖以及细胞因子诱导。由于针对4-1BB的不同激动性单克隆抗体在癌症、感染性疾病和自身免疫性疾病治疗中取得了令人鼓舞的结果，4-1BB已被认为是免疫治疗的一个有吸引力的靶点。在本研究中，利用Tomlinson I+J单链可变片段噬菌体展示文库，针对从4-1BB细胞外结构域设计的特定合成寡肽（肽I和肽II）进行筛选。经过五轮淘选，筛选出四个4-1BB特异性单链可变片段（PI.12、PI.42、PII.16和PII.29），它们在噬菌体酶联免疫吸附测定中对相关肽表现出特异性反应。所选克隆在大肠杆菌Rosetta-gami 2中成功表达，其表达通过蛋白质免疫印迹分析得到证实。酶联免疫吸附测定实验表明，这些抗体能够特异性识别4-1BB，与其他抗原无交叉反应。流式细胞术分析表明，单链可变片段与CCRF-CEM细胞上表达的4-1BB具有可接受的特异性结合，而与无关抗体未观察到结合。抗4-1BB单链可变片段增强了受刺激的CCRF-CEM细胞表面CD69的表达和白细胞介素-2的产生，这证实了所选单链可变片段的激动作用。本研究的数据为未来研究中涉及抗4-1BB单链可变片段生物学功能的进一步实验提供了理论依据。
