生物工程學(xué)報(bào) Chin J Biotech 2008, March 25; 24(3): 376-380
journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061
[email protected] . 2008 Institute of Microbiology, CAS & CSM, All rights reserved
人4-1BBL 胞外區(qū)/抗CD20 Fab’融合蛋白的構(gòu)建和表達(dá)
姜文國(guó)1, 熊冬生2, 劉芳2, 郭紅星2, 蘇曄2, 呂晶麗2, 楊純正2
1 濱州醫(yī)學(xué)院, 濱州 256603
2 中國(guó)協(xié)和醫(yī)學(xué)科大學(xué)/中國(guó)醫(yī)學(xué)科學(xué)院血液學(xué)研究所實(shí)驗(yàn)血液學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室, 天津 300020
摘 要: 研究共刺激分子4-1BBL 在腫瘤靶向治療方面的作用, 用PCR 和overlap PCR 方法構(gòu)建人4-1BBL 胞外區(qū)/抗CD20 Fab’融合蛋白表達(dá)載體, 并用雙脫氧終止法測(cè)定DNA 序列; 采用親和層析法純化該產(chǎn)物, 并用SDS-PAGE 和HPLC 鑒定純化產(chǎn)物; 采用玫瑰花環(huán)試驗(yàn)鑒定純化產(chǎn)物與靶細(xì)胞的結(jié)合活性,。DNA 序列測(cè)定結(jié)果表明: 人4-1BBL 胞外區(qū)/抗CD20 Fab’融合蛋白已構(gòu)建成功,。表達(dá)可溶性產(chǎn)物的產(chǎn)量達(dá)200 μg/L 以上, 純度較高, 具有與激活的Jurkat(4-1BBL+) 和Raji 細(xì)胞(CD20+)結(jié)合的活性,。這將為非何杰金氏淋巴瘤免疫治療,、靶向治療提供新的思路,。
關(guān)鍵詞: 4-1BBL, CD20, 靶向治療, 免疫治療
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
常規(guī)化療和放療選擇性差, 腫瘤細(xì)胞不能被完全清除,。殘存的腫瘤細(xì)胞, 即微小的殘留灶, 會(huì)造成治療失敗或者腫瘤復(fù)發(fā), 這將是免疫治療的靶標(biāo),。已經(jīng)有許多表達(dá)在惡性細(xì)胞上的腫瘤相關(guān)抗原(tumor-associated antigens, TAA)被證實(shí), 并且一些抗-TAA 的抗體已經(jīng)從實(shí)驗(yàn)室走向了臨床,?？?TAA的抗體不僅它們本身是靶向治療藥物, 而且還可以作為其他藥物和效應(yīng)細(xì)胞的靶向媒介,。
CD20 主要表達(dá)在前B 細(xì)胞和成熟B 細(xì)胞及95%以上的NHL 淋巴瘤細(xì)胞, 是可以用來(lái)作為腫瘤靶向免疫治療的抗原,。HI47(IgG3)是我所于1990 年研制成功的抗CD20 鼠源性單克隆抗體, 第四屆國(guó)際人類(lèi)白細(xì)胞分化抗原會(huì)議將HI47 命名為CD20+X,。CD20 單抗本身難以穿透實(shí)體腫瘤發(fā)揮殺傷作用, 而缺失恒定區(qū)的小型基因工程抗體雖然具有較好的穿透性, 但半衰期短且本身細(xì)胞毒作用低,。因此, 抗體片段與放射性同位素,、前藥(prodrug),、生物毒素及免疫效應(yīng)細(xì)胞形成偶聯(lián)物成為治療腫瘤特別是實(shí)體瘤的首選方案,。其中最有臨床應(yīng)用前景的是利用雙特異抗體使針對(duì)腫瘤抗原的抗體片段與免疫效應(yīng)細(xì)胞相連, 共同作用于腫瘤細(xì)胞, 實(shí)現(xiàn)腫瘤的靶向治療[1–3],。
近來(lái)的研究表明, 經(jīng)4-1BB/4-1BBL 信號(hào)途徑的協(xié)同刺激在許多模型顯示出抗腫瘤效應(yīng), 同時(shí)應(yīng)用4-1BBL 和CD3 單抗可代替專(zhuān)職抗原提呈細(xì)胞的刺激能力, 然而全身性的T 細(xì)胞激活將導(dǎo)致臨床副作用, 所以CD3/4-1BB 信號(hào)必須嚴(yán)格定位在腫瘤位點(diǎn),。為了特異性定向腫瘤細(xì)胞, 我們已成功構(gòu)建了抗CD3/抗CD20 Diabody 表達(dá)載體[2], 并證實(shí)抗CD3/抗CD20 Diabody 具有明顯的抑制腫瘤生長(zhǎng)的活性。為了進(jìn)一步提供腫瘤特異性的4-1BB 協(xié)同刺激, 我們構(gòu)建了人4-1BBL 胞外區(qū)基因表達(dá)載體pAYZ4-1BBL[4], 生物學(xué)活性研究發(fā)現(xiàn)它能使激活的T 細(xì)胞系IL-2 的釋放增加, 并使激活的T 細(xì)胞系凋亡減少1 倍以上, 在此基礎(chǔ)上我們研究并構(gòu)建了人4-1BBL 胞外區(qū)/抗CD20 Fab’融合蛋白表達(dá)載體,。
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