生物工程学报 Chin J Biotech 2008, March 25; 24(3): 376-380
journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061
cjb@im.ac.cn . 2008 Institute of Microbiology, CAS & CSM, All rights reserved
人4-1BBL 胞外区/抗CD20 Fab’融合蛋白的构建和表达
姜文国1, 熊冬生2, 刘芳2, 郭红星2, 苏晔2, 吕晶丽2, 杨纯正2
1 滨州医学院, 滨州 256603
2 中国协和医学科大学/中国医学科学院血液学研究所实验血液学国家重点实验室, 天津 300020
摘 要: 研究共刺激分子4-1BBL 在肿瘤靶向治疗方面的作用, 用PCR 和overlap PCR 方法构建人4-1BBL 胞外区/抗CD20 Fab’融合蛋白表达载体, 并用双脱氧终止法测定DNA 序列; 采用亲和层析法纯化该产物, 并用SDS-PAGE 和HPLC 鉴定纯化产物; 采用玫瑰花环试验鉴定纯化产物与靶细胞的结合活性。DNA 序列测定结果表明: 人4-1BBL 胞外区/抗CD20 Fab’融合蛋白已构建成功。表达可溶性产物的产量达200 μg/L 以上, 纯度较高, 具有与激活的Jurkat(4-1BBL+) 和Raji 细胞(CD20+)结合的活性。这将为非何杰金氏淋巴瘤免疫治疗、靶向治疗提供新的思路。
关键词: 4-1BBL, CD20, 靶向治疗, 免疫治疗
Study on the Construction and Expression of the Human 4-1BBL Extracellular Domain/anti-CD20 Fab’ Fusion Protein
Wenguo Jiang1, Dongsheng Xiong2, Fang Liu2, Hongxing Guo2, Ye Su2, Jingli Lü2, and Chunzheng Yang2
1 Binzhou Medical College, Binzhou 256603, China
2 The State Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS & PUMC, Tianjin 300020, China
Abstract: Several studies have demonstrated the role of 4-1BBL in T cell activation. Furthermore, enhanced 4-1BB/4-1BBL interaction has been shown to amplify T-cell-mediated antitumor immunity in several mouse models. However, when applied in humans, it was difficult to generate sufficient T cells ex vivo and whole cell vaccines to transfer back into patients. To overcome this difficulty, we have focused on producing the human 4-1BBL extracellular domain/anti-CD20 Fab’ fusion protein. In this report, PCR and overlap PCR were used to construct the human 4-1BBL extracellular domain/anti-CD20 Fab’ expression vector. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by SDS-PAGE and HPLC; its antigen binding activity was examined by rosetting assay. The data of DNA sequence showed that the human 4-1BBL extracellular domain / anti-CD20 Fab’ fusion protein was corrected. The fusion protein was recovered in high yield (up to 200 μg/mL) after E-taq purification. The fusion protein was capable of simultaneous binding to stimulated Jurkat cells and Raji cells as shown by cellular rosetting. In conclusion, the human 4-1BBL extracellular domain/anti-CD20 Fab’ fusion protein was induced to express in E. coli 16C9. The results of some biological activity experiments indicated that the fusion protein could bind to stimulated Jurkat cells and Raji cells. Furthermore, 4-1BBL-negative tumors can be converted into 4-1BBL-positive tumors by the fusion protein without the need for 4-1BBL gene transfer to the malignant cells.
Keywords: 4-1BB ligand, CD20, targeting therapy, cancer immunotherapy
常规化疗和放疗选择性差, 肿瘤细胞不能被完全清除。残存的肿瘤细胞, 即微小的残留灶, 会造成治疗失败或者肿瘤复发, 这将是免疫治疗的靶标。已经有许多表达在恶性细胞上的肿瘤相关抗原(tumor-associated antigens, TAA)被证实, 并且一些抗-TAA 的抗体已经从实验室走向了临床。抗-TAA的抗体不仅它们本身是靶向治疗药物, 而且还可以作为其他药物和效应细胞的靶向媒介。
CD20 主要表达在前B 细胞和成熟B 细胞及95%以上的NHL 淋巴瘤细胞, 是可以用来作为肿瘤靶向免疫治疗的抗原。HI47(IgG3)是我所于1990 年研制成功的抗CD20 鼠源性单克隆抗体, 第四届国际人类白细胞分化抗原会议将HI47 命名为CD20+X。CD20 单抗本身难以穿透实体肿瘤发挥杀伤作用, 而缺失恒定区的小型基因工程抗体虽然具有较好的穿透性, 但半衰期短且本身细胞毒作用低。因此, 抗体片段与放射性同位素、前药(prodrug)、生物毒素及免疫效应细胞形成偶联物成为治疗肿瘤特别是实体瘤的首选方案。其中最有临床应用前景的是利用双特异抗体使针对肿瘤抗原的抗体片段与免疫效应细胞相连, 共同作用于肿瘤细胞, 实现肿瘤的靶向治疗[1–3]。
近来的研究表明, 经4-1BB/4-1BBL 信号途径的协同刺激在许多模型显示出抗肿瘤效应, 同时应用4-1BBL 和CD3 单抗可代替专职抗原提呈细胞的刺激能力, 然而全身性的T 细胞激活将导致临床副作用, 所以CD3/4-1BB 信号必须严格定位在肿瘤位点。为了特异性定向肿瘤细胞, 我们已成功构建了抗CD3/抗CD20 Diabody 表达载体[2], 并证实抗CD3/抗CD20 Diabody 具有明显的抑制肿瘤生长的活性。为了进一步提供肿瘤特异性的4-1BB 协同刺激, 我们构建了人4-1BBL 胞外区基因表达载体pAYZ4-1BBL[4], 生物学活性研究发现它能使激活的T 细胞系IL-2 的释放增加, 并使激活的T 细胞系凋亡减少1 倍以上, 在此基础上我们研究并构建了人4-1BBL 胞外区/抗CD20 Fab’融合蛋白表达载体。
全文下载：人4-1BBL 胞外区/抗CD20 Fab’融合蛋白的构建和表达
更多全文请查看链接：http://journals.im.ac.cn
声明：本文由《生物工程学报》授权生物谷 www.bioon.com 网站发布，如需转载请直接与中国科学院微生物研究所期刊联合编辑部联系并支付相应费用，未经授权不得转载，若转载将付相应的法律责任。
