Article(id=1322906926526378757, tenantId=1146029695717560320, journalId=1235980733773295621, issueId=1322887411688919265, articleNumber=null, orderNo=null, doi=null, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=null, receivedDateStr=null, revisedDate=null, revisedDateStr=null, acceptedDate=1620230400000, acceptedDateStr=2021-05-06, onlineDate=1793335189706, onlineDateStr=2026-10-30, pubDate=1636905600000, pubDateStr=2021-11-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793335189706, onlineIssueDateStr=2026-10-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793335189706, creator=13701087609, updateTime=1793335189706, updator=13701087609, issue=Issue{id=1322887411688919265, tenantId=1146029695717560320, journalId=1235980733773295621, year='2021', volume='30', issue='21', pageStart='1921', pageEnd='2016', issueExtLink='null', onlineDate='null', pubDate='1636905600000', pubDateStr='2021-11-15', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1793330537006, creator='13701087609', updateTime=1793337443794, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1322916380919608076, tenantId=1146029695717560320, journalId=1235980733773295621, issueId=1322887411688919265, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1322916380919608077, tenantId=1146029695717560320, journalId=1235980733773295621, issueId=1322887411688919265, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1975, endPage=1981, ext={EN=ArticleExt(id=1322906926840951558, articleId=1322906926526378757, tenantId=1146029695717560320, journalId=1235980733773295621, language=EN, title=Preparation and preliminary characterization of anti-GPC3/NKG2D bispecific fusion protein, columnId=null, journalTitle=Chinese Journal of New Drugs, columnName=null, runingTitle=null, highlight=null, articleAbstract=
Objective: To construct bispecific fusion protein from Glypican-3 and NKG2D, and to primarily evaluate the cytolytic properties against human hepatocellular carcinoma cell line HepG2 in vitro.
Methods: Using genetic engineering technology, bispecific fusion protein FabGC33-MICA (human MHC class Ⅰ chain-related A, FabGM) expression vector was obtained by fusion the extracellular domain of MICA to heavy chain's C-terminal of the Fab form of GC33, and was used to transfect FreeStyleTM 293-F cells. FabGM fusion protein was purified from the cell supernatant by affinity chromatography. Octet QKe system (ForteBio) was applied to detect FabGM's affinity for its targets. Flow cytometry analysis was used to assess the avidity of FabGM fusion protein in HepG2 cells. The cytotoxicity of FabGM against HepG2 was monitored by IncuCyte Live Cell Imaging System. The secretion of interferon-γ (IFN-γ) by PBMCs was detected by ELISA assay.
Results: FabGM was successfully purified and confirmed selectively combined with GPC3 and NKG2D. The SDS-PAGE assay confirmed the predicted molecular. The Kd values of FabGM to target molecular GPC3 and NKG2D was 1.14 and 561 nmol·L-1 respectively. FabGM effectively mediated the targeted killing effect of PBMCs against HepG2. ELISA assay showed that IFN-γ level was significantly increased during the killing process compared with negative control.
Conclusion: Bispecific fusion protein FabGM effectively mediated PBMCs to kill HepG2 cells in vitro, providing experimental evidence for FabGM to become a candidate drug for hepatocellular carcinoma treatment.
, authors=Yun-hui LIU
1, 2, Chen GE
1, Qin-zhi XU
2, Ni-liang QIAN
2, Hui YANG
3, Ling-fei WAN
1, Xin-long YAN
1, Xin GAO
2, authorsList=Yun-hui LIU, Chen GE, Qin-zhi XU, Ni-liang QIAN, Hui YANG, Ling-fei WAN, Xin-long YAN, Xin GAO, authorCompany=null, correspAuthors=Xin-long YAN, Xin GAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1322906927189078791, articleId=1322906926526378757, tenantId=1146029695717560320, journalId=1235980733773295621, language=CN, title=抗GPC3/NKG2D双特异性融合蛋白的制备及其功能研究, columnId=1236330552693944345, journalTitle=中国新药杂志, columnName=生物医药前沿, runingTitle=null, highlight=null, articleAbstract=
目的： 构建靶向磷脂酰肌醇蛋白聚糖-3（glypican-3，GPC3）和NKG2D的双特异性融合蛋白，并初步评价其在体外对肝癌细胞的杀伤功能。
方法： 通过基因工程技术，将NKG2D的配体人主要组织相容性复合物Ⅰ类相关蛋白A（MHC class Ⅰ chain-related A，MICA）胞外区融合到Fab形式的抗人GPC3抗体GC33的重链末端，构建双特异性融合蛋白（Fab GC33-MICA，FabGM）表达载体；转染FreeStyleTM 293-F细胞，收集细胞培养上清，经亲和纯化获得FabGM蛋白；利用分子相互作用技术（Fortebio）和流式细胞术检测FabGM的靶向亲和力；细胞实时动态成像分析系统测定FabGM介导的外周血单个核细胞（PBMCs）对高表达GPC3的人肝癌细胞HepG2的杀伤效应，ELISA法检测杀伤过程中PBMCs分泌人干扰素-γ（hIFN-γ）情况。
结果： 本研究成功地表达并纯化了FabGM，明确FabGM可特异性结合GPC3和NKG2D。经检测FabGM能有效介导PBMCs靶向杀伤HepG2肝癌细胞，诱导PBMCs分泌IFN-γ。
结论： 双特异性融合蛋白FabGM能够在体外有效介导效应细胞PBMCs杀伤肝癌细胞HepG2，为FabGM成为肝细胞肝癌的潜在靶向治疗药物提供了实验依据。
, authors=刘蕴慧
1, 2, 葛晨
1, 徐勤枝
2, 钱尼良
2, 杨慧
3, 万令飞
1, 阎新龙
1, 高新
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1Faculty of Enviromental and Life Sciences, Beijing University of Technology, Beijing 100124, China
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刘蕴慧，女，硕士研究生，主要从事肿瘤生物学研究。E-mail：liuyunhui66@163.com。
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