免疫促凋亡分子e23sFv-TD-tBID的基因构建、表达及肿瘤靶向杀伤作用的研究
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摘要
重组免疫毒素是一类重要的肿瘤靶向性治疗药物。由具有细胞选择性作用的导向分子和细菌或植物毒素组成,它能特异性杀伤靶细胞,而不损伤正常组织细胞,可用于肿瘤、组织器官移植、自身免疫性疾病、HIV感染等免疫相关疾病。尽管重组免疫毒素具有广阔的应用前景,但是在临床应用中仍面临许多挑战,如免疫原性强和穿透性差,解决方法之一就是构建小分子人源化的重组免疫毒素。
Bid(BH3 -interacting death agonist)是Bcl-2家族BH3亚家族的一个成员,具有促凋亡活性。Bid蛋白只含BH3结构域,全长为195 aa,分子量22 KDa。Bid位于细胞浆,当其被活化的Caspase-8或其他蛋白酶切割后,去除N末端片段,产生的截短体片断(truncated Bid, tBid;15 kDa)从细胞浆转位到线粒体,主要通过控制细胞色素c(Cyt c)和其它促凋亡分子的释放来促进细胞凋亡。
HER2是癌基因erbB2/neu的表达产物,能传导生长信号而导致细胞的恶性增殖。HER2是目前公认的肿瘤细胞表面特异性标志分子,在乳腺癌等多种肿瘤细胞中高度表达,是一种理想的治疗靶点。
因此,以抗HER2单链抗体为导向分子,以人活性促凋亡分子tBid为毒性部分构建的重组免疫促凋亡分子是一种潜在的免疫原性低、穿透力强的抗肿瘤药物。
本研究中构建的重组免疫促凋亡分子由抗HER2单链抗体e23sFv和活性促凋亡分子tBid构成,白喉毒素转位结构域中含有Furin酶切位点的十肽(187 aa-196 aa)插入其间,连接导向分子和毒素分子。将重组基因克隆入原核表达载体pQE30,转化宿主菌,IPTG诱导表达,目的蛋白以包涵体形式存在。经亲和层析纯化、透析复性后,获得e23sFv-TD-tBID融合蛋白。进一步研究其生物学活性。
细胞ELISA、流式细胞术和间接免疫荧光结果显示,e23sFv-TD-tBID能特异地结合并内化入HER2阳性肿瘤细胞,对HER2阴性的细胞无此作用。MTT结果显示,e23sFv-TD-tBID能明显抑制HER2阳性肿瘤细胞的生长,对HER2高表达的SK-BR-3、SKOV-3细胞杀伤作用最强,这说明e23sFv-TD-tBID对靶细胞的杀伤作用和细胞表面HER2分子表达量密切相关。e23sFv-TD-tBID通过诱导凋亡来抑制肿瘤细胞生长。此外,我们还从乳腺癌病人的胸水中分离培养原代乳腺癌细胞,发现e23sFv-TD-tBID也可以结合并杀伤HER2阳性的原代乳腺癌细胞。
动物实验结果显示,单独使用e23sFv-TD-tBID治疗可以抑制荷瘤裸鼠肿瘤的生长,这种抑制作用具有剂量依赖性,在治疗期间未见明显的毒副作用。当e23sFv-TD-tBID与化疗药物联合使用时,抑瘤作用更加显著。
综上所述,本研究构建了重组免疫促凋亡分子e23sFv-TD-tBID,经原核表达、纯化、复性,获得e23sFv-TD-tBID融合蛋白。体外细胞学实验表明,e23sFv-TD-tBID能特异性结合并内化入HER2阳性肿瘤细胞诱导其凋亡,体内抑瘤实验表明,e23sFv-TD-tBID具有一定的抗肿瘤活性,与化疗药物联合使用作用更加显著,为进一步探讨其作为抗肿瘤药物的临床应用奠定了基础。
Recombinant immunotoxin is an important drug of targeted cancer therapy and is composed of a very potent protein toxin and a targeting moiety such as a recombinant antibody fragment or growth factor. Protein toxins commonly used include bacterial toxins such as PE and DT, and plant toxins such as ricin, PAP and saporin. The clinical efficacy of recombinant immunotoxins has been demonstrated in patients with malignant tumors. But the antitumor activity of recombinant immunotoxins in vivo is interfered by many factors, e.g, strong immunogenicity and poor penetration into tumors. One of methods is to develop new type of micromolecule recombinant immunotoxins to reduce their immunogenicity and improve their penetration into tumor cells.
Bid (BH3-interacting death agonist), one of the BH3 domain-only pro-apoptotic Bcl-2 family member, is recognized as an intracellular linker connecting the death receptors pathway and mitochondrial death machinery through caspase-8 activation. Full-length Bid,normally present in cytosol, is cleaved by activated caspase-8 or other proteases to form tBid (carboxyl-terminal region of Bid) which translocates to the mitochondria causing cytochrome c (Cyt c) and AIF release. Because Bid can link the extrinsic and intrinsic cell death pathways and amplify death receptor signaling, we believe tBid could be a potential candidate for cancer therapy.
HER2/neu (c-erbB2) proto-oncogene is located on chromosome 17q21 and encodes a Mr 185,000 transmembrane glycoprotein (p185HER2) that belongs to a subfamily of growth factor receptors having intrinsic tyrosine kinase activity. Amplification and overexpression of ERBB-2 is found in 25-30% of primary human breast cancers and is associated with a poor clinical outcome. Increased expression of ErbB2 is also observed in colon, prostate, lung, and gastric cancers, making it a popular therapeutic target for carcinomas. HER2/neu is an attractive target for immunotherapy.
In this study we constructed a noval human antitumor immunotBID molecule e23sFv-TD-tBID made up of anti-HER2 scFv (e23sFv) and active truncated tBID. A 10-residue spacer, Ala-Gly-Asn-Arg-Val-Arg-Arg-Ser-Val-Gly, containging the recognition site for protease furin from diphtheria toxin translocation domain was incorporated between the antibody fragment and the active truncated BID.
The recombinant gene was inserted into prokaryotic expression vector pQE30 and transformed into E.coli M15. The recombinant plasmid pQE30-e23sFv-TD-tBID expressed in inclsion body by the induction of IPTG. After purification by Ni-NTA chromatography and renaturation through dialysis, we get the fusion protein.
In vitro target-specific binding and cytotoxic activity of e23sFv-TD-tBID were show on a variety of HER2-positive cell lines and primary breast tumor cells. On binding to HER2, it was rapidly internalized into the target cells, after proteolytic cleavage, accessed directly to the cytosol. To determine the therapeutic effects of e23sFv-TD-tBID, it was given i.p. to immunodeficient mice bearing s.c. human breast carcinomas. The immunoproapoptotic protein either a single dose or combinations with chemotherapy significantly inhibited tumor growth with no toxic effect on mice. Our results suggest that e23sFv-TD-tBID has therapeutic potential for HER2-positive tumors and deserves further development.
Conclusion: We produced the fusion protein e23sFv-TD-tBID in E.coli. and described the characterization and antitumor activity in vitro and in vivo. These results show that this molecule may prove to be a valuble agent for immunotherapy of HER2-positive carcinomas.
Bid(BH3 -interacting death agonist)是Bcl-2家族BH3亚家族的一个成员,具有促凋亡活性。Bid蛋白只含BH3结构域,全长为195 aa,分子量22 KDa。Bid位于细胞浆,当其被活化的Caspase-8或其他蛋白酶切割后,去除N末端片段,产生的截短体片断(truncated Bid, tBid;15 kDa)从细胞浆转位到线粒体,主要通过控制细胞色素c(Cyt c)和其它促凋亡分子的释放来促进细胞凋亡。
HER2是癌基因erbB2/neu的表达产物,能传导生长信号而导致细胞的恶性增殖。HER2是目前公认的肿瘤细胞表面特异性标志分子,在乳腺癌等多种肿瘤细胞中高度表达,是一种理想的治疗靶点。
因此,以抗HER2单链抗体为导向分子,以人活性促凋亡分子tBid为毒性部分构建的重组免疫促凋亡分子是一种潜在的免疫原性低、穿透力强的抗肿瘤药物。
本研究中构建的重组免疫促凋亡分子由抗HER2单链抗体e23sFv和活性促凋亡分子tBid构成,白喉毒素转位结构域中含有Furin酶切位点的十肽(187 aa-196 aa)插入其间,连接导向分子和毒素分子。将重组基因克隆入原核表达载体pQE30,转化宿主菌,IPTG诱导表达,目的蛋白以包涵体形式存在。经亲和层析纯化、透析复性后,获得e23sFv-TD-tBID融合蛋白。进一步研究其生物学活性。
细胞ELISA、流式细胞术和间接免疫荧光结果显示,e23sFv-TD-tBID能特异地结合并内化入HER2阳性肿瘤细胞,对HER2阴性的细胞无此作用。MTT结果显示,e23sFv-TD-tBID能明显抑制HER2阳性肿瘤细胞的生长,对HER2高表达的SK-BR-3、SKOV-3细胞杀伤作用最强,这说明e23sFv-TD-tBID对靶细胞的杀伤作用和细胞表面HER2分子表达量密切相关。e23sFv-TD-tBID通过诱导凋亡来抑制肿瘤细胞生长。此外,我们还从乳腺癌病人的胸水中分离培养原代乳腺癌细胞,发现e23sFv-TD-tBID也可以结合并杀伤HER2阳性的原代乳腺癌细胞。
动物实验结果显示,单独使用e23sFv-TD-tBID治疗可以抑制荷瘤裸鼠肿瘤的生长,这种抑制作用具有剂量依赖性,在治疗期间未见明显的毒副作用。当e23sFv-TD-tBID与化疗药物联合使用时,抑瘤作用更加显著。
综上所述,本研究构建了重组免疫促凋亡分子e23sFv-TD-tBID,经原核表达、纯化、复性,获得e23sFv-TD-tBID融合蛋白。体外细胞学实验表明,e23sFv-TD-tBID能特异性结合并内化入HER2阳性肿瘤细胞诱导其凋亡,体内抑瘤实验表明,e23sFv-TD-tBID具有一定的抗肿瘤活性,与化疗药物联合使用作用更加显著,为进一步探讨其作为抗肿瘤药物的临床应用奠定了基础。
Recombinant immunotoxin is an important drug of targeted cancer therapy and is composed of a very potent protein toxin and a targeting moiety such as a recombinant antibody fragment or growth factor. Protein toxins commonly used include bacterial toxins such as PE and DT, and plant toxins such as ricin, PAP and saporin. The clinical efficacy of recombinant immunotoxins has been demonstrated in patients with malignant tumors. But the antitumor activity of recombinant immunotoxins in vivo is interfered by many factors, e.g, strong immunogenicity and poor penetration into tumors. One of methods is to develop new type of micromolecule recombinant immunotoxins to reduce their immunogenicity and improve their penetration into tumor cells.
Bid (BH3-interacting death agonist), one of the BH3 domain-only pro-apoptotic Bcl-2 family member, is recognized as an intracellular linker connecting the death receptors pathway and mitochondrial death machinery through caspase-8 activation. Full-length Bid,normally present in cytosol, is cleaved by activated caspase-8 or other proteases to form tBid (carboxyl-terminal region of Bid) which translocates to the mitochondria causing cytochrome c (Cyt c) and AIF release. Because Bid can link the extrinsic and intrinsic cell death pathways and amplify death receptor signaling, we believe tBid could be a potential candidate for cancer therapy.
HER2/neu (c-erbB2) proto-oncogene is located on chromosome 17q21 and encodes a Mr 185,000 transmembrane glycoprotein (p185HER2) that belongs to a subfamily of growth factor receptors having intrinsic tyrosine kinase activity. Amplification and overexpression of ERBB-2 is found in 25-30% of primary human breast cancers and is associated with a poor clinical outcome. Increased expression of ErbB2 is also observed in colon, prostate, lung, and gastric cancers, making it a popular therapeutic target for carcinomas. HER2/neu is an attractive target for immunotherapy.
In this study we constructed a noval human antitumor immunotBID molecule e23sFv-TD-tBID made up of anti-HER2 scFv (e23sFv) and active truncated tBID. A 10-residue spacer, Ala-Gly-Asn-Arg-Val-Arg-Arg-Ser-Val-Gly, containging the recognition site for protease furin from diphtheria toxin translocation domain was incorporated between the antibody fragment and the active truncated BID.
The recombinant gene was inserted into prokaryotic expression vector pQE30 and transformed into E.coli M15. The recombinant plasmid pQE30-e23sFv-TD-tBID expressed in inclsion body by the induction of IPTG. After purification by Ni-NTA chromatography and renaturation through dialysis, we get the fusion protein.
In vitro target-specific binding and cytotoxic activity of e23sFv-TD-tBID were show on a variety of HER2-positive cell lines and primary breast tumor cells. On binding to HER2, it was rapidly internalized into the target cells, after proteolytic cleavage, accessed directly to the cytosol. To determine the therapeutic effects of e23sFv-TD-tBID, it was given i.p. to immunodeficient mice bearing s.c. human breast carcinomas. The immunoproapoptotic protein either a single dose or combinations with chemotherapy significantly inhibited tumor growth with no toxic effect on mice. Our results suggest that e23sFv-TD-tBID has therapeutic potential for HER2-positive tumors and deserves further development.
Conclusion: We produced the fusion protein e23sFv-TD-tBID in E.coli. and described the characterization and antitumor activity in vitro and in vivo. These results show that this molecule may prove to be a valuble agent for immunotherapy of HER2-positive carcinomas.
引文
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85. Owens.R.J. and Young.R.J. The genetic engineering of monoclonal antibodies. J Immunol Methods. 1994, 168: 149-165.
86. Worm.A. and Pluekthun.A. Mutual stabilization of VL and VH in single-chain antibody fragments investigated with mutants engineered for stability. Biochemistry.1998, 22: 13120-13127.
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90. Duebel S. Reconstitution of human pancreatic Rnase from two separate fragments fused to Different single chain antibody fragments: On the way to binary immunotoxins. Tumor Targeting, 1999, 4: 37-46.
91. Susanna M R, Hennie RH, HarryMM, et al. Humanization of immunotoxins. PNAS, 1992, 89: 3165-3169.
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93. Azar Y, Lorberboum G H. GnRH2Bik/ Bax/ Bak chimeric proteins target and kill adenocarcinoma cells: the general use of pro-apoptotic proteins of the Bcl-2 family as novel killing components of targeting chimeric proteins. Apoptosis, 2000, 5(6): 531-542.
94. Jia LT, Zhang LH, Yu CJ, et al. Specific tumouricidal activity of a secreted pro-apoptotic protein consisting of HER2 antibody and constitutively active caspase-3. Cancer Res, 2003, 63: 3257–62.
95. Xu YM, Wang LF, Jia LT, et al. A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumours. J Immunol, 2004, 173: 61–7.
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97. Yu CJ, Jia LT, Meng YL, et al. Selective pro-apoptotic activity of a secreted recombinant antibody/AIF fusion protein in carcinomas overexpressing HER2. Gene Ther, 2006, 13: 313–20.
98. Xiu-Chun Qiu, Yan-Ming Xu, Fang Wang, et al. Single-chain antibody/activated BID chimeric protein effctively suppresses HER2-positive tumor growth. Mol Cancer Ther, 2008, 7(7): 1890-1899.
99. Stephen P. Ethier, Michael L. Mahacek, et al. Differential isolation of normal luminal mammary epithelial cells and breast cancer cells from primary and metastatic sites using selective media. Cancer Res, 1993, 53: 627-635.
100. Antia GOYAl, Janendra K.BATRA. Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins. Biochem. J., 2000, 345: 247-254.