抗EGFR单链抗体与力达霉素融合蛋白的构建及其抗肿瘤活性研究
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摘要
表皮生长因子受体(epidermal growth factor receptor,EGFR)是一种酪氨酸激酶受体,是ErbB家族四个成员之一,在多种上皮来源的肿瘤中都呈过表达。EGFR及其配体是细胞信号传导系统的一部分,其信号传导网络在肿瘤的形成和发展过程中起重要作用,因此EGFR是肿瘤治疗中一个很重要的靶分子。
抗体导向治疗是以抗体为载体,将药物导向肿瘤病灶,提高药物疗效,降低药物的副作用。单链抗体(single-chain Fv,scFv)是一种分子较小的基因工程抗体片段,其分子量小,免疫原性弱,组织穿透能力强,较易穿透细胞外间隙到达肿瘤内部。但是由于单链抗体片段缺乏Fc段,丧失完整抗体的效应功能,难以杀伤靶细胞,因此需要使用“弹头”分子,包括高效的药物、毒素、细胞因子等。
力达霉素(lidamycin,LDM)是由球孢链霉菌(Streptiomyces globisporus)产生的烯二炔类抗生素,是迄今报道过的对肿瘤细胞杀伤作用最强的大分子肽类抗肿瘤抗生素。LDM的分子由烯二炔结构的发色团(AE)和辅基蛋白(LDP)构成,两者可进行拆分和重组,因此已成为构建新型抗体靶向药物理想的“弹头”药物。
本研究主要目的是利用噬菌体展示技术构建噬菌体单链抗体库,筛选EGFR高亲和力单链抗体。利用基因重组技术构建表达单链抗体与力达霉素辅基蛋白的融合蛋白ER(Fv-LDP),并与力达霉素发色团组装成强化融合蛋白ER(Fv-LDP-AE),研究其对EGFR高表达肿瘤细胞的体外杀伤作用以及体内抑瘤活性。
1.抗EGFR噬菌体单链抗体库的构建筛选及单链抗体可溶表达
本研究用EGFR高表达的人鳞状上皮癌A431细胞免疫小鼠,从脾细胞提取mRNA,利用RT-PCR扩增抗体重链可变区(VH)和轻链可变区(VL)基因。利用重叠延伸反应(SOE-PCR)将VH、VL用连接肽(Gly_4Ser)_3进行连接,导入噬菌粒pCANTAB5E,电击转化E.coli TG1细胞,用辅助噬菌体M13K07感染,获得库容为2.5×10~7的噬菌体单链抗体库。MvaⅠ内切酶酶切图谱显示抗体库具有多样性。用纯化的EGFR为靶抗原对噬菌体抗体库进行5轮富集筛选,得到次级抗体库6F-10。ELISA方法鉴定各单克隆与EGFR蛋白结合能力,在随机挑取的48个克隆中,有45个克隆产生的噬菌体抗体能与EGFR发生特异性结合,阳性率为93%。取阳性值最高的克隆感染E.coli HB2151,IPTG诱导抗EGFR单链抗体ER(Fv)的表达。约27kDa大小的ER(Fv)以可溶形式存在于细胞质及细胞周质中,并可分泌至上清。测序结果显示,ER(Fv)基因序列全长768bp,编码256个氨基酸。VH为与小鼠Ig同源的重链可变区基因,VL为κ型轻链可变区基因;VH和VL均由3个抗原互补决定区和4个框架区构成。免疫印迹和细胞免疫荧光证明可溶性ER(Fv)可分别与纯化的EGFR抗原以及细胞表面的EGFR发生特异性结合。抗EGFR单链抗体ER(Fv)的获得,为研制靶向EGFR的抗体药物与研究生物治疗提供导向载体分子。
2.抗EGFR单链抗体与力达霉素辅基蛋白融合蛋白ER(Fv-LDP)的构建及活性分析
利用基因重组技术将筛选出来的EGFR高亲和力单链抗体片段基因连接到含有力达霉素辅基蛋白基因的重组质粒pET-30(a)-LDP,构建重组质粒pET-30(a)-scFv-LDP,转化到E.coli BL21(DE3) star~(TM)中,IPTG诱导带有组氨酸标签肽(His-tag)的融合蛋白的表达。融合蛋白分子量约为38kDa,主要以包涵体形式存在。Ni离子亲和柱纯化融合蛋白,蛋白收获量约为5mg/L。ELISA检测及细胞免疫荧光显示融合蛋白与纯EGFR抗原的相对亲和力为8.0×10~(-9)M,对EGFR高表达的肿瘤细胞的亲和力明显高于低表达的正常细胞。流式细胞仪检测结果表明融合蛋白可引起细胞周期的G2/M期阻滞,且这种抑制作用存在着一定的剂量依赖关系。细胞增殖实验证明融合蛋白可抑制肿瘤细胞的增殖,其对EGFR高表达的A431和A549细胞的杀伤作用明显强于EGFR低表达的HEK293细胞。体内抑瘤实验表明融合蛋白能够显著抑制人鳞状上皮癌A431裸鼠移植瘤的生长,且毒副作用较小。融合蛋白0.5 mg/kg和5mg/kg 2个剂量组35天的抑瘤率分别为51.4%和67.5%,与对照组和LDP组相比有显著差异。
3.抗EGFR单链抗体与力达霉素强化融合蛋白ER(Fv-LDP-AE)的制备及活性分析
将抗EGFR单链抗体与力达霉素辅基蛋白的融合蛋白与力达霉素活性发色团组装,得到强化融合蛋白。流式细胞仪检测结果表明强化融合蛋白可引起细胞周期的G2/M期阻滞,且这种抑制作用存在着一定的剂量依赖关系。Hoechst染色显示强化融合蛋白可以诱导肿瘤细胞的凋亡。细胞增殖实验证明强化融合蛋白对肿瘤细胞具有强杀伤作用,且强化融合蛋白作用要明显强于单纯的LDM,其作用于EGFR高表达的A431和A549细胞的IC_(50)值分别是LDM的1/7和1/10。体内抑瘤实验表明强化融合蛋白对人鳞状上皮癌A431裸鼠移植瘤具有明显的抑制作用,且毒副作用较LDM小。强化融合蛋白0.2mg/kg和0.3mg/kg2个剂量组30天抑瘤率分别为75.4%和81.3%,与LDM组相比存在显著差异。强化融合蛋白可以引起细胞周期G2/M阻滞,对EGFR高表达的肿瘤细胞有较强的体外杀伤活性,对人鳞状上皮癌A431裸鼠移植瘤有显著的抑制作用,有望成为一种具有良好应用前景的抗体靶向药物。
The epidermal growth factor receptor(EGFR) is a tyrosine kinase receptor of the ErbB family that is overexpressed in many epithelial tumors.EGFR and its ligands serve as a part of intracellular signaling pathways,which play an important role in the formation and the development of tumors.It has been recognized that EGFR is a promising target for cancer therapy.
Single chain Fv(scFv) is an engineered antibody fragment.Compared to the complete antibody,scFv shows lower molecular weight,lower immunogenicity,and strong penetration capability,which can penetrate into solid tumor deeply.Due to the lack of Fc,scFv can not kill target tumor cells,thus a "warhead" molecule including drugs,toxins,and cytokines is needed.
Lidamycin(LDM),a macromolecular peptide antibiotic produced by Streptomyces globisporus,shows extremely potent cytotoxicity against tumor cells.LDM consists of an active enedinye chomophore(AE) and an apoprotein(LDP),which can be separated and reconstituted without losing its activity.Therefore,LDM is regarded as an ideal "warhead" molecule for antibody targeted drugs.
In this study,phage display was used to select strong affinity scFv for EGFR.Fusion protein ER(Fv-LDP) was prepared and energized fusion protein ER(Fv-LDP-AE) was reconstituted.The potent cytotoxicity of fusion protein and its energized protein was observed.
1.Construction and screening of phage antibody libraries against EGFR and soluble expression of ER(Fv)
Balb/c mice were immunized by human epidermoid carcinoma A431 cells,and total RNA of the splenic cells was extracted.VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker,then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E.The phagemides containing scFv were transformed into electro-competent E.coli TG1 cells.The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified by ELISA.A total of 48 clones from the library was selected randomly and 45 clones were identified positive. After infecting E.coli HB2151 cells with one positive clone,soluble recombinant antibodies about 27 kDa were produced and located in the periplasm and the supernatant. The result of sequencing showed that ER(Fv) gene was 768bp,which encoded 256 amino acid residues.VH and VL including 3 CDRs and 4 FRs respectively were all homologous to mouse Ig.The soluble ER(Fv) showed specific binding activity to purified EGFR and the EGFR located in carcinoma cell membrane.The successful preparation of ER(Fv) will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.
2.Construction of fusion protein ER(Fv-LDP) and the study of its activity
ScFv gene was ligated into the plasmid pET-30a(+)-LDP,then the recombinant plasmid pET-30a(+)-scFv-LDP was transformed into competent E.coli BL21(DE3) star~(TM) cells.Fusion protein with His-tag was produced in the form of inclusion after IPTG inducing.Finally about 5 mg of purified protein was obtained from 1 L of culture medium.ELISA and immunofluorescence assays showed that ER(Fv-LDP) had strong affinity for A431 and A549 cells that overexpress EGFR.FACS ananlysis of cell cycle showed that the cells were arrested in G2/M phase after ER(Fv-LDP) treatment,and the degree of arrest was decided by the dose of fusion protein.The cytotoxicity of ER(Fv-LDP) was checked by MTT.ER(Fv-LDP) showed potent cytotoxicity against tumor cells overexpressing EGFR.Human epidermoid carcinoma A431 xenograft in nude mice was used to investigate inhibitory effects of the fusion protein in vivo. ER(Fv-LDP) showed strong antitumor activity and less side effects.The inhibition rates of ER(Fv-LDP) at dose of 0.5 mg/kg and 5 mg/kg were 54.1%and 67.5%respectively.
3.Preparation of energized fusion protein ER(Fv-LDP-AE) and the study of its antitumor activity
The energized fusion protein ER(Fv-LDP-AE) was prepared by the reconstitution of ER(Fv-LDP-AE) and AE.FACS ananlysis of cell cycle showed that cells were also arrested in G2/M phase after ER(Fv-LDP-AE) treatment,and the degree of arrest was decided by protein dose.Apoptosis of tumor cells treated with ER(Fv-LDP-AE) was observed after Hoechst dying.It is observed in MTT that ER(Fv-LDP-AE) had stronger cytotoxicity than LDM.The IC_(50) value of ER(Fv-LDP-AE) was only 1/7 of that of LDM for A431 cells and 1/10 for A549 cells respectively.In vivo,ER(Fv-LDP-AE) showed stronger antitumor activity than LDM.The inhibition rates of ER(Fv-LDP-AE) at dose of 0.2 mg/kg and 0.3 mg/kg were 75.4%and 85.3%,while that of LDM at 0.05 mg/kg was 64.1%respectively.The successful preparation of energized protein ER(Fv-LDP-AE) will provide a promising EGFR targeted drug for the therapy of cancer.
抗体导向治疗是以抗体为载体,将药物导向肿瘤病灶,提高药物疗效,降低药物的副作用。单链抗体(single-chain Fv,scFv)是一种分子较小的基因工程抗体片段,其分子量小,免疫原性弱,组织穿透能力强,较易穿透细胞外间隙到达肿瘤内部。但是由于单链抗体片段缺乏Fc段,丧失完整抗体的效应功能,难以杀伤靶细胞,因此需要使用“弹头”分子,包括高效的药物、毒素、细胞因子等。
力达霉素(lidamycin,LDM)是由球孢链霉菌(Streptiomyces globisporus)产生的烯二炔类抗生素,是迄今报道过的对肿瘤细胞杀伤作用最强的大分子肽类抗肿瘤抗生素。LDM的分子由烯二炔结构的发色团(AE)和辅基蛋白(LDP)构成,两者可进行拆分和重组,因此已成为构建新型抗体靶向药物理想的“弹头”药物。
本研究主要目的是利用噬菌体展示技术构建噬菌体单链抗体库,筛选EGFR高亲和力单链抗体。利用基因重组技术构建表达单链抗体与力达霉素辅基蛋白的融合蛋白ER(Fv-LDP),并与力达霉素发色团组装成强化融合蛋白ER(Fv-LDP-AE),研究其对EGFR高表达肿瘤细胞的体外杀伤作用以及体内抑瘤活性。
1.抗EGFR噬菌体单链抗体库的构建筛选及单链抗体可溶表达
本研究用EGFR高表达的人鳞状上皮癌A431细胞免疫小鼠,从脾细胞提取mRNA,利用RT-PCR扩增抗体重链可变区(VH)和轻链可变区(VL)基因。利用重叠延伸反应(SOE-PCR)将VH、VL用连接肽(Gly_4Ser)_3进行连接,导入噬菌粒pCANTAB5E,电击转化E.coli TG1细胞,用辅助噬菌体M13K07感染,获得库容为2.5×10~7的噬菌体单链抗体库。MvaⅠ内切酶酶切图谱显示抗体库具有多样性。用纯化的EGFR为靶抗原对噬菌体抗体库进行5轮富集筛选,得到次级抗体库6F-10。ELISA方法鉴定各单克隆与EGFR蛋白结合能力,在随机挑取的48个克隆中,有45个克隆产生的噬菌体抗体能与EGFR发生特异性结合,阳性率为93%。取阳性值最高的克隆感染E.coli HB2151,IPTG诱导抗EGFR单链抗体ER(Fv)的表达。约27kDa大小的ER(Fv)以可溶形式存在于细胞质及细胞周质中,并可分泌至上清。测序结果显示,ER(Fv)基因序列全长768bp,编码256个氨基酸。VH为与小鼠Ig同源的重链可变区基因,VL为κ型轻链可变区基因;VH和VL均由3个抗原互补决定区和4个框架区构成。免疫印迹和细胞免疫荧光证明可溶性ER(Fv)可分别与纯化的EGFR抗原以及细胞表面的EGFR发生特异性结合。抗EGFR单链抗体ER(Fv)的获得,为研制靶向EGFR的抗体药物与研究生物治疗提供导向载体分子。
2.抗EGFR单链抗体与力达霉素辅基蛋白融合蛋白ER(Fv-LDP)的构建及活性分析
利用基因重组技术将筛选出来的EGFR高亲和力单链抗体片段基因连接到含有力达霉素辅基蛋白基因的重组质粒pET-30(a)-LDP,构建重组质粒pET-30(a)-scFv-LDP,转化到E.coli BL21(DE3) star~(TM)中,IPTG诱导带有组氨酸标签肽(His-tag)的融合蛋白的表达。融合蛋白分子量约为38kDa,主要以包涵体形式存在。Ni离子亲和柱纯化融合蛋白,蛋白收获量约为5mg/L。ELISA检测及细胞免疫荧光显示融合蛋白与纯EGFR抗原的相对亲和力为8.0×10~(-9)M,对EGFR高表达的肿瘤细胞的亲和力明显高于低表达的正常细胞。流式细胞仪检测结果表明融合蛋白可引起细胞周期的G2/M期阻滞,且这种抑制作用存在着一定的剂量依赖关系。细胞增殖实验证明融合蛋白可抑制肿瘤细胞的增殖,其对EGFR高表达的A431和A549细胞的杀伤作用明显强于EGFR低表达的HEK293细胞。体内抑瘤实验表明融合蛋白能够显著抑制人鳞状上皮癌A431裸鼠移植瘤的生长,且毒副作用较小。融合蛋白0.5 mg/kg和5mg/kg 2个剂量组35天的抑瘤率分别为51.4%和67.5%,与对照组和LDP组相比有显著差异。
3.抗EGFR单链抗体与力达霉素强化融合蛋白ER(Fv-LDP-AE)的制备及活性分析
将抗EGFR单链抗体与力达霉素辅基蛋白的融合蛋白与力达霉素活性发色团组装,得到强化融合蛋白。流式细胞仪检测结果表明强化融合蛋白可引起细胞周期的G2/M期阻滞,且这种抑制作用存在着一定的剂量依赖关系。Hoechst染色显示强化融合蛋白可以诱导肿瘤细胞的凋亡。细胞增殖实验证明强化融合蛋白对肿瘤细胞具有强杀伤作用,且强化融合蛋白作用要明显强于单纯的LDM,其作用于EGFR高表达的A431和A549细胞的IC_(50)值分别是LDM的1/7和1/10。体内抑瘤实验表明强化融合蛋白对人鳞状上皮癌A431裸鼠移植瘤具有明显的抑制作用,且毒副作用较LDM小。强化融合蛋白0.2mg/kg和0.3mg/kg2个剂量组30天抑瘤率分别为75.4%和81.3%,与LDM组相比存在显著差异。强化融合蛋白可以引起细胞周期G2/M阻滞,对EGFR高表达的肿瘤细胞有较强的体外杀伤活性,对人鳞状上皮癌A431裸鼠移植瘤有显著的抑制作用,有望成为一种具有良好应用前景的抗体靶向药物。
The epidermal growth factor receptor(EGFR) is a tyrosine kinase receptor of the ErbB family that is overexpressed in many epithelial tumors.EGFR and its ligands serve as a part of intracellular signaling pathways,which play an important role in the formation and the development of tumors.It has been recognized that EGFR is a promising target for cancer therapy.
Single chain Fv(scFv) is an engineered antibody fragment.Compared to the complete antibody,scFv shows lower molecular weight,lower immunogenicity,and strong penetration capability,which can penetrate into solid tumor deeply.Due to the lack of Fc,scFv can not kill target tumor cells,thus a "warhead" molecule including drugs,toxins,and cytokines is needed.
Lidamycin(LDM),a macromolecular peptide antibiotic produced by Streptomyces globisporus,shows extremely potent cytotoxicity against tumor cells.LDM consists of an active enedinye chomophore(AE) and an apoprotein(LDP),which can be separated and reconstituted without losing its activity.Therefore,LDM is regarded as an ideal "warhead" molecule for antibody targeted drugs.
In this study,phage display was used to select strong affinity scFv for EGFR.Fusion protein ER(Fv-LDP) was prepared and energized fusion protein ER(Fv-LDP-AE) was reconstituted.The potent cytotoxicity of fusion protein and its energized protein was observed.
1.Construction and screening of phage antibody libraries against EGFR and soluble expression of ER(Fv)
Balb/c mice were immunized by human epidermoid carcinoma A431 cells,and total RNA of the splenic cells was extracted.VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker,then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E.The phagemides containing scFv were transformed into electro-competent E.coli TG1 cells.The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified by ELISA.A total of 48 clones from the library was selected randomly and 45 clones were identified positive. After infecting E.coli HB2151 cells with one positive clone,soluble recombinant antibodies about 27 kDa were produced and located in the periplasm and the supernatant. The result of sequencing showed that ER(Fv) gene was 768bp,which encoded 256 amino acid residues.VH and VL including 3 CDRs and 4 FRs respectively were all homologous to mouse Ig.The soluble ER(Fv) showed specific binding activity to purified EGFR and the EGFR located in carcinoma cell membrane.The successful preparation of ER(Fv) will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.
2.Construction of fusion protein ER(Fv-LDP) and the study of its activity
ScFv gene was ligated into the plasmid pET-30a(+)-LDP,then the recombinant plasmid pET-30a(+)-scFv-LDP was transformed into competent E.coli BL21(DE3) star~(TM) cells.Fusion protein with His-tag was produced in the form of inclusion after IPTG inducing.Finally about 5 mg of purified protein was obtained from 1 L of culture medium.ELISA and immunofluorescence assays showed that ER(Fv-LDP) had strong affinity for A431 and A549 cells that overexpress EGFR.FACS ananlysis of cell cycle showed that the cells were arrested in G2/M phase after ER(Fv-LDP) treatment,and the degree of arrest was decided by the dose of fusion protein.The cytotoxicity of ER(Fv-LDP) was checked by MTT.ER(Fv-LDP) showed potent cytotoxicity against tumor cells overexpressing EGFR.Human epidermoid carcinoma A431 xenograft in nude mice was used to investigate inhibitory effects of the fusion protein in vivo. ER(Fv-LDP) showed strong antitumor activity and less side effects.The inhibition rates of ER(Fv-LDP) at dose of 0.5 mg/kg and 5 mg/kg were 54.1%and 67.5%respectively.
3.Preparation of energized fusion protein ER(Fv-LDP-AE) and the study of its antitumor activity
The energized fusion protein ER(Fv-LDP-AE) was prepared by the reconstitution of ER(Fv-LDP-AE) and AE.FACS ananlysis of cell cycle showed that cells were also arrested in G2/M phase after ER(Fv-LDP-AE) treatment,and the degree of arrest was decided by protein dose.Apoptosis of tumor cells treated with ER(Fv-LDP-AE) was observed after Hoechst dying.It is observed in MTT that ER(Fv-LDP-AE) had stronger cytotoxicity than LDM.The IC_(50) value of ER(Fv-LDP-AE) was only 1/7 of that of LDM for A431 cells and 1/10 for A549 cells respectively.In vivo,ER(Fv-LDP-AE) showed stronger antitumor activity than LDM.The inhibition rates of ER(Fv-LDP-AE) at dose of 0.2 mg/kg and 0.3 mg/kg were 75.4%and 85.3%,while that of LDM at 0.05 mg/kg was 64.1%respectively.The successful preparation of energized protein ER(Fv-LDP-AE) will provide a promising EGFR targeted drug for the therapy of cancer.
引文
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