噬菌体抗体库技术筛选抗VEGF165单克隆抗体
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摘要
目的:获得抗血管内皮生长因子165(VEGF165)单克隆抗体,并对其功能进行初步验证。方法:利用噬菌体抗体库展示技术筛选与VEGF165结合的噬菌体克隆并测序,以测序正确的阳性克隆质粒为模板,PCR扩增抗体的轻重链可变区基因,并克隆至哺乳动物细胞表达载体中,构建全抗体表达载体;将全抗体表达载体转染293E细胞,收取培养细胞上清,利用Protein A亲和纯化抗体;通过结合ELISA、表面等离子共振检测抗体的亲和力,以人脐静脉内皮细胞(HUVEC)为模型验证抗体功能。结果:经过噬菌体抗体库展示技术筛出1个与VEGF165特异性结合的抗体序列VG2;293E细胞表达了VG2全抗体蛋白,SDS-PAGE显示VG2抗体纯度较高;BIAcore检测结果表明该抗体具有较高亲和力(KD=0.56 nmol/L),竞争抑制ELISA结果表明VG2抗体能抑制VEGF与VEGF受体(VEGFR)的结合(IC50为1.470μg/mL),进一步实验结果表明VG2能够抑制VEGF引起的HUVEC增殖。结论:制备了靶向VEGF165的全人源单克隆抗体VG2,该抗体具有较高的亲和力,能阻断VEGF165/VEGFR2的结合,并抑制HUVEC的增殖,可以作为潜在药物应用于肿瘤治疗。
Objective: To obtain monoclonal antibodies against vascular endothelial growth factor 165(VEGF165 and to verify their function preliminarily. Methods: The phage clones that bind to VEGF165 were screened by phage antibody library display technology and sequenced. The positive clones were verified and used as templates,and the light and heavy chain variable region genes of the antibodies were amplified by PCR and cloned into mammalian cell expression vectors. The full antibody expression vector was transfected into 293 E cells, and the cultured cell supernatant was collected, and the antibody was affinity-purified using Protein A. The affinity of the antibody was detected by binding ELISA, surface plasmon resonance. Finally, human umbilical vein endothelial cells(HUVEC) were used as a model to verify the function of antibody. Results: An antibody sequence specifically binding to VEGF165 was screened by phage antibody library display technology and was named as VG2. VG2 full-antibody was successfully expressed in 293 E cells, and SDS-PAGE experiments showed that VG2 antibody was highly purified. The BIAcore test showed that the antibody had a high affinity, KD=0.56 nmol/L. The competitive inhibition ELISA showed that VG2 antibody inhibited the binding of VEGF to VEGF receptor(VEGFR) with an IC50 of 1.470 μg/mL. Further experimental showed that VG2 can inhibit the proliferation of HUVEC induced by VEGF 165. Conclusion: We have prepared a fully human monoclonal antibody VG2 targeting VEGF165 with high affinity, which can block the binding of VEGF/VEGFR2 and inhibit the proliferation of HUVEC. It might be used as a potential drug for tumor therapy.
Objective: To obtain monoclonal antibodies against vascular endothelial growth factor 165(VEGF165 and to verify their function preliminarily. Methods: The phage clones that bind to VEGF165 were screened by phage antibody library display technology and sequenced. The positive clones were verified and used as templates,and the light and heavy chain variable region genes of the antibodies were amplified by PCR and cloned into mammalian cell expression vectors. The full antibody expression vector was transfected into 293 E cells, and the cultured cell supernatant was collected, and the antibody was affinity-purified using Protein A. The affinity of the antibody was detected by binding ELISA, surface plasmon resonance. Finally, human umbilical vein endothelial cells(HUVEC) were used as a model to verify the function of antibody. Results: An antibody sequence specifically binding to VEGF165 was screened by phage antibody library display technology and was named as VG2. VG2 full-antibody was successfully expressed in 293 E cells, and SDS-PAGE experiments showed that VG2 antibody was highly purified. The BIAcore test showed that the antibody had a high affinity, KD=0.56 nmol/L. The competitive inhibition ELISA showed that VG2 antibody inhibited the binding of VEGF to VEGF receptor(VEGFR) with an IC50 of 1.470 μg/mL. Further experimental showed that VG2 can inhibit the proliferation of HUVEC induced by VEGF 165. Conclusion: We have prepared a fully human monoclonal antibody VG2 targeting VEGF165 with high affinity, which can block the binding of VEGF/VEGFR2 and inhibit the proliferation of HUVEC. It might be used as a potential drug for tumor therapy.
引文
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[5]Mccafferty J,Griffiths A D,Winter G,et al.Phage antibodies:filamentous phage displaying antibody variable domains[J].Nature,1990,348(6301):552-554.
[6]Chellappan D K,Leng K H,Jia L J,et al.The role of bevacizumab on tumour angiogenesis and in the management of gynaecological cancers:a review[J].Biomed Pharmacother,2018,102:1127-1144.
[7]Fernando N H,Hurwitz H I.Inhibition of vascular endothelial growth factor in the treatment of colorectal cancer[J].Semin Oncol,2003,30(3,Suppl 6):39-50.
[8]Li X,Li W,Hou L.A trial-based cost-effectiveness analysis of Bevacizumab and chemotherapy versus chemotherapy alone for advanced nonsquamous non-smallcell lung cancer in China[J].Value Health Reg Issues,2019,18:1-7.
[9]Mehany S A,Shawkat A M,Sayed M F,et al.Role of Avastin in management of central serous chorioretinopathy[J].Saudi J Ophthalmol,2010,24(3):69-75.
[2]徐梦龙,谭树华.噬菌体展示技术在抗体药物研发中的应用[J].黑龙江科技信息,2017,(13):72.
[3]侯雪莹,胡卓伟,崔冰.噬菌体展示技术在药物研发中的应用[J].药学学报,2018,53(8):1280-1289.
[4]Smith G P.Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J].Science,1985,228(4705):1315-1317.
[5]Mccafferty J,Griffiths A D,Winter G,et al.Phage antibodies:filamentous phage displaying antibody variable domains[J].Nature,1990,348(6301):552-554.
[6]Chellappan D K,Leng K H,Jia L J,et al.The role of bevacizumab on tumour angiogenesis and in the management of gynaecological cancers:a review[J].Biomed Pharmacother,2018,102:1127-1144.
[7]Fernando N H,Hurwitz H I.Inhibition of vascular endothelial growth factor in the treatment of colorectal cancer[J].Semin Oncol,2003,30(3,Suppl 6):39-50.
[8]Li X,Li W,Hou L.A trial-based cost-effectiveness analysis of Bevacizumab and chemotherapy versus chemotherapy alone for advanced nonsquamous non-smallcell lung cancer in China[J].Value Health Reg Issues,2019,18:1-7.
[9]Mehany S A,Shawkat A M,Sayed M F,et al.Role of Avastin in management of central serous chorioretinopathy[J].Saudi J Ophthalmol,2010,24(3):69-75.