整合PD-L1单链抗体的融合蛋白制备及其抗肿瘤活性的验证
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摘要
目的:利用酵母表面递呈技术,分泌表达抗PD-L1单链抗体(sc Fv),纯化后得到能特异性结合PD-L1抗原的小分子抗体片段(sc Fv)。根据单链抗体基因序列,合成sc Fv抗体基因序列。选用sc Fv-mFc融合蛋白的方式,并采用p Fuse真核表达载体来表达此sc Fv-mFc融合蛋白,研究其对肺腺癌细胞(A549)的亲和力和体内外抑制作用。方法:采用基因工程的方法构建重组质粒p Fuse-scFv,通过真核转染至293F(人胚肾细胞),使用无血清Pro 293a-CDM培养72 h,收集培养液中分泌的融合蛋白,运用免疫组化检测sc Fv-mFc融合蛋白与肿瘤细胞的结合;流式细胞术分析融合蛋白和肿瘤细胞的亲和力; ADCC(抗体介导的细胞毒实验)测定融合蛋白对肿瘤细胞体外杀伤作用;利用接种肺腺癌细胞的荷瘤小鼠对融合蛋白的体内抗肿瘤效应进行研究。结果:通过重组质粒转染至293F细胞的方法,sc Fv-mFc融合蛋白被分泌到无血清的培养液中;免疫组化和流式结果显示,融合蛋白与表面高表达PD-L1蛋白的肿瘤细胞有较强的结合能力; ADCC实验结果示融合蛋白在体外对肿瘤细胞的杀伤作用;荷瘤小鼠实验结果示融合蛋白对肿瘤的生长起到抑制作用,在5 mg/kg的药物剂量下,荷瘤小鼠瘤体体积平均增长率从14. 90%降低至3. 72%,两独立样本t检验P<0. 05,差异具有统计学意义。结论:成功制备了含单链抗体的融合蛋白,其对A549细胞具有良好的结合能力,在体内外均对肿瘤细胞的增殖产生抑制作用,为研制靶向抗肿瘤药物提供实验室基础依据。
Objective: Using yeast surface presentation technology,secreted anti-PD-L1 single-chain antibody fragment( sc Fv),then purify the sc Fv that specifically binds PD-L1 antigen. The sc Fv antibody gene sequence was synthesized based on the single chain antibody gene sequence. We express this sc Fv-mFc protein by using p Fuse eukaryotic expression vector to study its affinity and in vitro and in vivo inhibition of lung adenocarcinoma cells( A549). Methods: Recombinant plasmid p Fuse-scFv was constructed by gene engineering. The recombinant plasmid p Fuse-scFv was transfected into 293 F( human embryonic kidney cells) and cultured in serum-free Pro293 a-CDM for 72 hours,then the fusion protein was collected,and use the Rapid Protein Liquid Phase Separation and Purification System to purify the sc Fv-mFc fusion protein. Then the fusion protein and the tumor cells were detected by immunohistochemistry; the affinity of fusion protein and tumor cells was analyzed by flow cytometry; ADCC was used to determine the proliferation of tumor cells in vitro. The nude mice inoculated with lung adenocarcinoma cells,and use the fusion protein to verify its anti-tumor effect in vivo. Results: sc Fv-mFc fusion protein was secreted into serum-free culture medium by recombinant plasmid transfection into the 293 F cells; immunohistochemistry and flow cytometry showed that the fusion protein was highly expressed with the surface of PD-L1 protein;ADCC showed that the fusion protein inhibited the proliferation of tumor cells in vitro; the results of tumor-bearing mice showed that the fusion protein inhibited the growth of the tumor. At the dose of 5 mg/kg,The tumor volume growth rate decreased from 14. 90% to3. 72%,the two independent samples t test P<0. 05,the difference was statistically significant. Conclusion: The fusion protein containing single chain antibody was successfully prepared,which had good binding ability to A549 cells and inhibited the proliferation of tumor cells in vitro and in vivo,and provided the laboratory basis for the development of targeted anti-tumor drugs.
Objective: Using yeast surface presentation technology,secreted anti-PD-L1 single-chain antibody fragment( sc Fv),then purify the sc Fv that specifically binds PD-L1 antigen. The sc Fv antibody gene sequence was synthesized based on the single chain antibody gene sequence. We express this sc Fv-mFc protein by using p Fuse eukaryotic expression vector to study its affinity and in vitro and in vivo inhibition of lung adenocarcinoma cells( A549). Methods: Recombinant plasmid p Fuse-scFv was constructed by gene engineering. The recombinant plasmid p Fuse-scFv was transfected into 293 F( human embryonic kidney cells) and cultured in serum-free Pro293 a-CDM for 72 hours,then the fusion protein was collected,and use the Rapid Protein Liquid Phase Separation and Purification System to purify the sc Fv-mFc fusion protein. Then the fusion protein and the tumor cells were detected by immunohistochemistry; the affinity of fusion protein and tumor cells was analyzed by flow cytometry; ADCC was used to determine the proliferation of tumor cells in vitro. The nude mice inoculated with lung adenocarcinoma cells,and use the fusion protein to verify its anti-tumor effect in vivo. Results: sc Fv-mFc fusion protein was secreted into serum-free culture medium by recombinant plasmid transfection into the 293 F cells; immunohistochemistry and flow cytometry showed that the fusion protein was highly expressed with the surface of PD-L1 protein;ADCC showed that the fusion protein inhibited the proliferation of tumor cells in vitro; the results of tumor-bearing mice showed that the fusion protein inhibited the growth of the tumor. At the dose of 5 mg/kg,The tumor volume growth rate decreased from 14. 90% to3. 72%,the two independent samples t test P<0. 05,the difference was statistically significant. Conclusion: The fusion protein containing single chain antibody was successfully prepared,which had good binding ability to A549 cells and inhibited the proliferation of tumor cells in vitro and in vivo,and provided the laboratory basis for the development of targeted anti-tumor drugs.
引文
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[7]Pardoll DM.The blockade of immune checkpoints in cancer immunotherapy[J].Nat Rev Cancer,2012,12(4):252-264.
[8]Ni L,Dong C.New B7 Family Checkpoints in Human Cancer[J].Mol Cancer Ther,2017,16(7):1203-1211.
[9]Jacquin-Porretaz C,Nardin C,Pazenat E,et al.Adverse effects of Immune checkpoints inhibitors used to treat melanoma and other cancer[J].Press Med,2017,4982(17):30271-30273.
[10]Cho YH,Kim MS,Chung HS,et al.Novel immunotherapy in matastatic renal cell carcinoma[J].Investig Clin Urol,2017,58(4):220-227.
[11]Kim EY,Kim A,Kim SY,et al.MYC expression correlates with PD-L1 in non-small cell lung cancer[J].Lung Cancer,2017,110(6):63-67.
[12]Kawai S,Yamazaki K.Immune checkpoints in Colorectal Cancer[J].Gan To Kagaku Ryoho,2016,43(11):1347-1351.
[13]Herbst RS,Soria JC,Kowanetz M.Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients[J].Nature,2014,515(7528):563-567.
[14]Sfanos KS,Bruno TC,Meeker AK.Human prostate-infiltrating CD8+Tlymphocytes are oligoclonal and PD-1[J].Prostate,2009,69(15):1694-1703.
[15]Soliman H,Khalil F,Antonia S.PD-L1 expression is increased in a subset of basal type breast cancer cells[J].PLoS One,2014,9(2):e88557.
[16]Liu Y,Carlsson R,Ambjorn M.PD-L1 Expression by neurons nearby tumors indicates better prognosis in gliblastoma patients[J].J Neurosci,2013,33(35):14231-14245.
[17]韩岩梅,曹雪涛.准确把握肿瘤免疫治疗的发展趋势,促进我国肿瘤免疫治疗规范健康发展[J].中国肿瘤生物治疗杂志,2017,24(1):2-5.Han YM,Cao XT.Grasp the trends of tumor immunotherapy accurately to promote its orderly and healthy development in China[J].China J Cancer Biother,2017,24(1):2-5.
[18]Couzin-Frankel J.Breakthrough of the year 2013.Cancer immunotherapy[J].Science,2013,342(6165):1432-1433.
[19]Luo L,Shu R,Wu A.Nanomaterial-based cancer immunotherapy[J].J Mat Chem B,2017,5(28):5517-5531.