Article(id=1210517367105385436, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210517366081975259, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2022-0121, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1643126400000, receivedDateStr=2022-01-26, revisedDate=1648137600000, revisedDateStr=2022-03-25, acceptedDate=null, acceptedDateStr=null, onlineDate=1766539430643, onlineDateStr=2025-12-24, pubDate=1668182400000, pubDateStr=2022-11-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1766539430643, onlineIssueDateStr=2025-12-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1766539430643, creator=13701087609, updateTime=1766539430643, updator=13701087609, issue=Issue{id=1210517366081975259, tenantId=1146029695717560320, journalId=1189982191388893191, year='2022', volume='57', issue='11', pageStart='3259', pageEnd='3450', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1766539430399, creator=13701087609, updateTime=1766539608198, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1210518111875363690, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210517366081975259, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1210518111875363691, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210517366081975259, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=3310, endPage=3315, ext={EN=ArticleExt(id=1210517368002966498, articleId=1210517367105385436, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Preparation of anti-human TIGIT monoclonal antibody and preliminary study of its biological activity, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

T cell immune receptor with Ig and ITIM domains (TIGIT), a promising new target in cancer immunotherapy, plays a critical role in limiting adaptive and innate immunity against tumors. The extracellular domain of human TIGIT was used to immune BALB/c mice, and a new anti-human TIGIT chimeric antibody (c7D3) was developed. The mice in this study were used in accordance with the international guidelines for the care and use of laboratory animals, and the animal study was approved by the Institutional Animal Ethics Committee of AbMax Biotechnology. The biological activity of c7D3 was studied. The results showed that c7D3 exhibited high affinity for TIGIT and effectively inhibited the interaction between TIGIT and its ligands. Cell-based assays indicated that c7D3 induced strong luciferase signaling in TIGIT/CD155 signaling reporter assay and enhanced cytokine secretion in a T cell stimulation assay. The data showed that c7D3 has high binding affinity and excellent blocking bioactivity, supporting the further advancement for therapeutic application.

, correspAuthors=Hai-bin WANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2022 Acta Pharmaceutica Sinica. All rights reserved., 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, authorCompany=null, fund=null, authors=null, authorsList=Zhen-hua WU, Na LI, Juan CHEN, Mei-zhu JIANG, Yao CHEN, Xiao-fen MEI, Hai-bin WANG), CN=ArticleExt(id=1210517370691514405, articleId=1210517367105385436, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=抗TIGIT单克隆抗体的制备及体外生物学活性的初步研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT) 通过多种机制抑制固有和适应性免疫, 是肿瘤免疫治疗的新型靶点。本研究利用重组人源TIGIT胞外区蛋白免疫BALB/c小鼠(本研究中的小鼠按照国际实验动物护理和使用准则进行使用, 并经委托单位京天成生物技术(北京) 有限公司实验动物伦理委员会批准), 分离并提取小鼠脾淋巴细胞常规融合, 成功筛选得到抗TIGIT单克隆抗体c7D3, 并初步研究了其生物学活性。结果表明, 抗体c7D3能高亲和地结合TIGIT, 并能有效阻断TIGIT与配体的结合。细胞功能实验证实, c7D3能激活T细胞, 在TIGIT/CD155报告基因实验中促进荧光素酶表达, 提高人外周血单个核细胞分泌细胞因子的能力。本研究表明, 抗体c7D3具有高亲和力和良好的生物学活性, 有望开发成为新的肿瘤免疫治疗的抗体药物。

, correspAuthors=王海彬, authorNote=null, correspAuthorsNote=
*王海彬, Tel: 86-576-89007929, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2022, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=fl2YUH3VdFMvUDTuyNyFvA==, magXml=iCDecyOwq6cpXNh9Fi7ZgQ==, pdfUrl=null, pdf=EASkVFjtiZQGJTnSSJtUsA==, pdfFileSize=1417344, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=xk12qujf6CkhIUsmZE3+2g==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=vgIar55GQeLM7vaDspkwUQ==, mapNumber=null, authorCompany=null, fund=null, authors=

#共同第一作者

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Front Immunol, 2022, 13: 828319., articleTitle=An Fc-competent anti-human TIGIT blocking antibody ociperlimab (BGBA1217) elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1210517370888646709, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, xref=null, ext=[AuthorCompanyExt(id=1210517370897035319, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, companyId=1210517370888646709, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=BioRay Pharmaceutical Co., Ltd., Taizhou 318000, China), AuthorCompanyExt(id=1210517370901229624, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, companyId=1210517370888646709, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=浙江博锐生物制药有限公司, 浙江 台州 318000)])], figs=[ArticleFig(id=1210517375540130103, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, language=EN, label=null, caption=null, figureFileSmall=5sEU3405B2nOtBXLKdVUlg==, figureFileBig=xk12qujf6CkhIUsmZE3+2g==, tableContent=null), ArticleFig(id=1210517375619821887, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, language=CN, label=Figure 1, caption= The binding activity of hybridoma supernatants to TIGIT (A) and the blocking activity towards TIGIT/CD155 interaction (B) were determined by ELISA. 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M: Protein marker (kDa); Lane 1: c7D3 under reducing condition , figureFileSmall=MFccDHTTX/lQAFE9QvAkVA==, figureFileBig=BIYKFqJIRmG/TfM6E3ggYA==, tableContent=null), ArticleFig(id=1210517376077001047, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, language=EN, label=null, caption=null, figureFileSmall=qBApwIz06SMkEqpkCaU+nQ==, figureFileBig=UrcTiU1NbZ9VL0+CuNfvgQ==, tableContent=null), ArticleFig(id=1210517376177664351, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, language=CN, label=Figure 3, caption= The binding activities of c7D3. A: Analysis of TIGIT expression in 293T-TIGIT cell line by flow cytometry; B: Binding to recombinant TIGIT was analyzed by ELISA; C: Binding to 293T-TIGIT cell line was analyzed by flow cytometry. <i>n</i> = 3, mean ± SEM , figureFileSmall=qBApwIz06SMkEqpkCaU+nQ==, figureFileBig=UrcTiU1NbZ9VL0+CuNfvgQ==, tableContent=null), ArticleFig(id=1210517376286716261, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, language=EN, label=null, caption=null, figureFileSmall=mUf7Cz51I/QUrwbl/b+CPw==, figureFileBig=MHrrydzVVcREGUiJARxBbw==, tableContent=null), ArticleFig(id=1210517376408351088, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, language=CN, label=Figure 4, caption= The blocking activities of c7D3 towards TIGIT/CD155 interaction (A) and TIGIT/CD112 interaction (B) were determined by competition inhibition ELISA. <i>n</i> = 3, mean ± SEM , figureFileSmall=mUf7Cz51I/QUrwbl/b+CPw==, figureFileBig=MHrrydzVVcREGUiJARxBbw==, tableContent=null), ArticleFig(id=1210517376509014391, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, language=EN, label=null, caption=null, figureFileSmall=eHFzvu+bYbO1LtGzBasQyg==, figureFileBig=FaxaJ23kiiOP7E06DJ6EKA==, tableContent=null), ArticleFig(id=1210517376626454909, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517367105385436, language=CN, label=Figure 5, caption= Functional assay of c7D3. 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抗TIGIT单克隆抗体的制备及体外生物学活性的初步研究
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吴振华 # , 李娜 # , 陈娟 , 蒋美珠 , 陈瑶 , 梅小芬 , 王海彬 *
药学学报 | 研究论文 2022,57(11): 3310-3315
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药学学报 | 研究论文 2022, 57(11): 3310-3315
抗TIGIT单克隆抗体的制备及体外生物学活性的初步研究
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吴振华#, 李娜#, 陈娟, 蒋美珠, 陈瑶, 梅小芬, 王海彬*
作者信息
  • 浙江博锐生物制药有限公司, 浙江 台州 318000

通讯作者:

*王海彬, Tel: 86-576-89007929, E-mail:
Preparation of anti-human TIGIT monoclonal antibody and preliminary study of its biological activity
Zhen-hua WU, Na LI, Juan CHEN, Mei-zhu JIANG, Yao CHEN, Xiao-fen MEI, Hai-bin WANG*
Affiliations
  • BioRay Pharmaceutical Co., Ltd., Taizhou 318000, China
出版时间: 2022-11-12 doi: 10.16438/j.0513-4870.2022-0121
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T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT) 通过多种机制抑制固有和适应性免疫, 是肿瘤免疫治疗的新型靶点。本研究利用重组人源TIGIT胞外区蛋白免疫BALB/c小鼠(本研究中的小鼠按照国际实验动物护理和使用准则进行使用, 并经委托单位京天成生物技术(北京) 有限公司实验动物伦理委员会批准), 分离并提取小鼠脾淋巴细胞常规融合, 成功筛选得到抗TIGIT单克隆抗体c7D3, 并初步研究了其生物学活性。结果表明, 抗体c7D3能高亲和地结合TIGIT, 并能有效阻断TIGIT与配体的结合。细胞功能实验证实, c7D3能激活T细胞, 在TIGIT/CD155报告基因实验中促进荧光素酶表达, 提高人外周血单个核细胞分泌细胞因子的能力。本研究表明, 抗体c7D3具有高亲和力和良好的生物学活性, 有望开发成为新的肿瘤免疫治疗的抗体药物。

TIGIT  /  杂交瘤  /  单克隆抗体  /  嵌合抗体  /  生物学活性

T cell immune receptor with Ig and ITIM domains (TIGIT), a promising new target in cancer immunotherapy, plays a critical role in limiting adaptive and innate immunity against tumors. The extracellular domain of human TIGIT was used to immune BALB/c mice, and a new anti-human TIGIT chimeric antibody (c7D3) was developed. The mice in this study were used in accordance with the international guidelines for the care and use of laboratory animals, and the animal study was approved by the Institutional Animal Ethics Committee of AbMax Biotechnology. The biological activity of c7D3 was studied. The results showed that c7D3 exhibited high affinity for TIGIT and effectively inhibited the interaction between TIGIT and its ligands. Cell-based assays indicated that c7D3 induced strong luciferase signaling in TIGIT/CD155 signaling reporter assay and enhanced cytokine secretion in a T cell stimulation assay. The data showed that c7D3 has high binding affinity and excellent blocking bioactivity, supporting the further advancement for therapeutic application.

TIGIT  /  hybridoma  /  monoclonal antibody  /  chimeric antibody  /  bioactivity
吴振华, 李娜, 陈娟, 蒋美珠, 陈瑶, 梅小芬, 王海彬. 抗TIGIT单克隆抗体的制备及体外生物学活性的初步研究. 药学学报, 2022 , 57 (11) : 3310 -3315 . DOI: 10.16438/j.0513-4870.2022-0121
Zhen-hua WU, Na LI, Juan CHEN, Mei-zhu JIANG, Yao CHEN, Xiao-fen MEI, Hai-bin WANG. Preparation of anti-human TIGIT monoclonal antibody and preliminary study of its biological activity[J]. Acta Pharmaceutica Sinica, 2022 , 57 (11) : 3310 -3315 . DOI: 10.16438/j.0513-4870.2022-0121
近年来, 肿瘤免疫治疗彻底改变了晚期恶性肿瘤的临床治疗方式, 成为继手术、放疗和化疗后又一重要手段[1, 2]。以程序性死亡受体1/程序性死亡配体1 (PD-1/PD-L1)、细胞毒性T淋巴细胞相关蛋白-4 (CTLA-4) 等免疫检查点抑制剂为代表的免疫疗法可以阻断免疫抑制信号, 从而促进T细胞的激活和对肿瘤的免疫应答[3]。但是, 目前免疫疗法存在响应率不高和耐药性的问题, 表明存在其他免疫耐受机制帮助肿瘤逃脱免疫监控[4, 5]
T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT) 是一种免疫共抑制受体, 属免疫球蛋白超家族成员, 主要表达于效应T细胞、记忆T细胞、调节T细胞(Treg)、自然杀伤(NK) 细胞中[6, 7]。TIGIT目前已知的配体有CD155和CD112, 其主要在树突状细胞(DC)、巨噬细胞、T细胞等免疫细胞表面表达, 此外, 在很多肿瘤细胞上也有高表达[8, 9]。TIGIT信号通路在肿瘤细胞逃脱免疫监视中发挥了重要作用。TIGIT与CD155或CD112的结合可减弱DC细胞的抗原呈递功能, 或直接向T细胞和NK细胞内部传递抑制性信号[10]。当TIGIT在Treg上表达时, TIGIT信号通路增强Treg的抑制功能, 从而抑制多种免疫细胞的活性[11, 12]。研究表明, 阻断TIGIT信号通路可增强效应T细胞和NK细胞活性, 促进免疫细胞表达干扰素-γ (IFN-γ)[13, 14]。在小鼠肿瘤模型中靶向TIGIT单抗可减缓或抑制肿瘤的生长, 临床上抗TIGIT单抗与其他免疫检查点抑制剂的联用可提高患者的响应率, 并降低疾病风险[15-17]。因此, 特异性的抗TIGIT单抗可增强抗肿瘤免疫应答, 是实现肿瘤免疫治疗的有效策略。
为开发新型高功效的TIGIT抗体, 本研究利用重组人胞外区TIGIT蛋白免疫BALB/c小鼠, 筛选得到一株特异性抗TIGIT单克隆抗体, 并初步研究了其生物学活性, 为后续进一步的开发奠定了基础。
细胞系  293T细胞来源于美国典型培养物保藏中心(ATCC); 表达TIGIT的慢病毒上清来源于赛业生物科技有限公司; CHO-S细胞来源于Thermo Fisher公司; TIGIT效应细胞、CD155 aAPC/CHO-K1细胞来源于金斯瑞生物科技股份有限公司; 人外周血单个核细胞(PBMC) 来源于澳赛尔斯生物技术有限公司(Allcells)。
主要材料和试剂  BALB/c小鼠, 4周龄, 购于北京维通利华实验动物技术有限公司, 动物许可证号: SYXK (京) 2017-0033; 胎牛血清(FBS)、磷酸缓冲盐溶液(PBS)、DMEM培养基购自Gibco公司; Hybri-Max HAT培养基补充物(HAT)、弗氏完全和不完全佐剂购自Sigma公司; PEG2000购自Roche公司, CD155、TIGIT蛋白、生物素标记TIGIT蛋白购自北京百普赛斯生物科技股份有限公司(Acrobiosystems); BSA购自生工生物工程(上海) 股份有限公司; TMB购自湖州英创生物科技有限公司; HRP标记的山羊抗人IgG、FITC标记的山羊抗人IgG、HRP标记的链霉亲和素购自Abcam公司; 对照抗体22G2、IgG1购自泰州市百英生物科技有限公司; 镍柱购自Solarbio公司; 抗PD-L1抗体为本公司保存; 白细胞介素2 (IL-2) 和IFN-γ检测试剂盒购自Invitrogen公司。
动物免疫及细胞融合  委托京天成生物技术(北京) 有限公司开展, 以制备的100 μg胞外区TIGIT蛋白(氨基酸Met22~Pro141) 为免疫原, 加弗氏完全佐剂乳化后, 背部多点皮下注射免疫BALB/c小鼠, 并分离成功免疫的小鼠脾脏淋巴细胞, 按1∶1比例与对数生长期的小鼠骨髓瘤细胞SP2/0进行细胞融合, 用含15% FBS、1×HAT的DMEM选择性培养基培养杂交瘤细胞。动物实验符合国际实验动物护理和使用准则, 并经京天成生物技术(北京) 有限公司实验动物伦理委员会批准。
细胞株的筛选及抗体纯化  通过结合和竞争ELISA实验筛选杂交瘤上清液, 将筛选出的阳性杂交瘤用有限稀释法克隆化培养, 经多次克隆化培养后, 挑选出稳定的杂交瘤细胞株。小鼠腹腔注射杂交瘤细胞制备腹水, 腹水经饱和硫酸铵沉淀和亲和层析纯化制备鼠抗人TIGIT单克隆抗体。
轻重链可变区测序及抗体表达  按照常规方法对筛选到的阳性杂交瘤细胞株进行RNA提取并逆转录为cDNA, 并对抗体基因序列进行序列确定, 得到7D3的重链可变区和轻链可变区序列。将7D3的重链、轻链可变区分别和人源抗体重链IgG1恒定区、人源抗体轻链kappa恒定区连接, 克隆到载体pBRex-3中并转染至CHO-S细胞, 在37 ℃、5% CO2的摇床上培养约7天后收集上清液, 用protein A纯化, 得到嵌合抗体c7D3。将纯化后的抗体通过SDS-PAGE凝胶电泳和SEC-HPLC法检测。
ELISA检测抗体与人TIGIT的结合活性  将人TIGIT以0.5 μg·mL-1的浓度稀释到PBS中, 每孔100 μL包被微孔板4 ℃过夜。弃包被液, PBST洗板后, 用5% BSA-PBST封闭液37 ℃封闭1 h; 洗板后, 每孔加入起始浓度133.33 nmol·L-1、4倍梯度稀释的抗TIGIT抗体100 μL, 并在37 ℃孵育1 h。反应完毕洗板, 每孔加入1∶10 000稀释HRP标记的山羊抗人IgG 100 μL, 37 ℃反应1 h。洗板后加入TMB溶液, 室温避光反应10 min后, 加入1 mol·L-1 H2SO4终止反应。置于酶标仪上450 nm波长检测吸光度, 并用GraphPad Prism 7软件进行四参数曲线拟合, 计算半数有效浓度(EC50) 值。
293T-TIGIT细胞株构建  取表达TIGIT的慢病毒上清感染293T细胞, 48 h后, 进行加压筛选。2周后, 流式细胞术检测TIGIT的表达。
流式细胞术检测抗体与细胞膜表达的TIGIT结合活性  收集对数期生长的293T-TIGIT细胞, 以2% BSA-PBS离心洗涤细胞2次, 计数后, 每管1×105个分装, 加入起始浓度13.3 nmol·L-1、5倍梯度稀释的c7D3抗体于4 ℃反应30 min。洗涤细胞2次后, 加入1∶100稀释的FITC标记的山羊抗人IgG, 4 ℃避光反应30 min。洗涤细胞2次后, 用流式细胞仪检测。
竞争ELISA法检测抗体对TIGIT与CD155结合的抑制  将CD155以PBS稀释至3 μg·mL-1, 每孔100 μL加入酶标板中4 ℃包被过夜。PBST洗板后加入2% BSA-PBST封闭液, 37 ℃封闭1.5 h。将起始浓度133.33 nmol·L-1抗体进行4倍稀释, 与1 μg·mL-1生物素标记的TIGIT每孔各50 μL加入板上, 37 ℃孵育1 h。洗板后每孔加入1∶1 000稀释的HRP标记的链霉亲和素100 μL, 37 ℃反应1 h。洗板后, 每孔加入TMB溶液100 μL, 室温避光反应30 min后, 以1 mol·L-1 H2SO4终止反应, 酶标仪检测450 nm波长吸光度, 并用GraphPad Prism 7软件进行四参数曲线拟合, 计算半数抑制浓度(IC50) 值。
竞争ELISA法检测抗体对TIGIT与CD112结合的抑制  将CD112以PBS稀释至4 μg·mL-1, 每孔100 μL加入酶标板中4 ℃包被过夜。PBST洗板后加入2% BSA-PBST封闭液, 37 ℃封闭1.5 h。将起始浓度133.33 nmol·L-1的抗体进行4倍稀释, 与4 μg·mL-1生物素标记的TIGIT每孔各50 μL加入板上, 37 ℃孵育1 h。洗板后每孔加入1∶1 000稀释的HRP标记的链霉亲和素100 μL, 37 ℃反应1 h。洗板后, 每孔加入TMB溶液100 μL, 室温避光反应30 min后, 以1 mol·L-1 H2SO4终止反应, 酶标仪检测450 nm波长吸光度, 并用GraphPad Prism 7软件进行四参数曲线拟合, 计算IC50值。
抗体抑制TIGIT/CD155报告基因活性  将CD155 aAPC/CHO-K1细胞接种到96孔细胞培养板, 培养箱中孵育16~20 h, 然后加入TIGIT效应细胞和c7D3抗体, 培养板置于培养箱中孵育约6 h, 加入荧光素酶底物, 酶标仪上读取化学发光值。依据相对化学发光信号值(RLU) 和最终检测浓度的对应关系, 用GraphPad Prism 7软件进行四参数曲线拟合建立相应的量效曲线图。
抗体激活PBMC细胞释放细胞因子  将CD155以PBS稀释至1 μg·mL-1的浓度, 每孔100 μL加至细胞培养板中4 ℃包被过夜; 弃上清, 每孔加入1×105个PBMC细胞、50 ng·mL-1金黄色葡萄球菌肠毒素B (SEB), 及梯度稀释的抗体c7D3或等量的同型IgG1空白对照抗体或c7D3抗体与PD-L1抗体的组合, 置于37 ℃、5% CO2培养箱孵育4天。收集细胞培养上清, ELISA检测IL-2和IFN-γ的浓度。
统计学分析  实验数据均采用GraphPad Prism 7处理, 表示为mean ± SEM。两组间的差异性比较采用Student's t检验分析。
以重组人TIGIT蛋白胞外区为免疫原免疫小鼠, 采用细胞融合方式构建并获得杂交瘤细胞, ELISA检测筛选出10个阳性杂交瘤克隆(图 1A)。竞争ELISA法检测这10个杂交瘤细胞株阻断TIGIT与CD155结合的抑制率, 筛选出抑制率最强的单克隆细胞株7D3 (图 1B)。制备并纯化鼠抗人单克隆抗体7D3, 将对应杂交瘤细胞株进行轻重链可变区测序, 并与人源抗体重链IgG1恒定区、人源抗体轻链kappa恒定区连接, 表达并纯化嵌合抗体c7D3。SDS-PAGE电泳检测, 表明c7D3抗体纯度 > 95% (图 2A), 通过SEC-HPLC测定抗体纯度为97.1% (图 2B)。
通过慢病毒转染构建表达TIGIT的293T细胞株, 以抗体22G2检测细胞株表达, 流式检测结果见图 3A, 与293T细胞相比, 293T-TIGIT细胞表达TIGIT的阳性细胞达到90%以上, 表明293T-TIGIT细胞可以稳定表达TIGIT。
采用ELISA和流式细胞术的方法检测单克隆抗体c7D3与TIGIT的结合活性。包被人TIGIT蛋白, 检测不同浓度的c7D3与TIGIT的结合, 结果见图 3B, c7D3与TIGIT结合的EC50值为0.104 nmol·L-1, 对照抗体22G2与TIGIT结合的EC50值为0.491 nmol·L-1, 表明c7D3与TIGIT具有较高的结合活性, 且其结合活性强于22G2。以构建好的293T-TIGIT细胞株, 检测单克隆抗体c7D3与膜表达TIGIT的结合, 如图 3C所示, 表明c7D3能特异性地结合膜表达的TIGIT。
分别包被CD155和CD112蛋白, 采用竞争ELISA法检测单克隆抗体c7D3对TIGIT与CD155、CD112结合的阻断作用, 结果见图 4AB, 在抗体阻断TIGIT与CD155结合实验中, c7D3和22G2的IC50分别为3.338和8.869 nmol·L-1; 抗体阻断TIGIT与CD112结合实验中, c7D3和22G2的IC50分别为18.90和43.39 nmol·L-1。结果表明, c7D3抗体能有效阻断TIGIT/CD155和TIGIT/CD112的结合, 且与对照抗体22G2相比, 阻断作用更强。
TIGIT/CD155报告基因法以表达人源TIGIT和萤光素酶报告基因的Jurkat T细胞为效应细胞, 以表达人源CD155及T细胞受体(TCR) 激活蛋白的CHO-K1细胞为抗原呈递细胞。当两种细胞共培养时, TIGIT与其配体CD155相互作用, 抑制IL2启动子调控的荧光素酶表达, 因此, 在实验体系中引入抗TIGIT阻断性抗体后, 可与效应细胞表达的TIGIT相结合, 阻断TIGIT与CD155的相互作用, 解除抑制信号, 并激活TCR信号途径和IL2启动子, 促进荧光素酶的表达, 并催化底物产生化学发光信号。TIGIT/CD155报告基因实验结果见图 5A, 与IgG1对照抗体相比, c7D3诱导了化学发光信号值的增加, 表明c7D3能有效抑制TIGIT与其配体的结合, 并激活T细胞活化信号通路。
96孔培养板上包被CD155, 加入SEB体外刺激PBMC分泌细胞因子, 通过检测IL-2和IFN-γ水平评价抗体对人原代T淋巴细胞功能的影响。结果如图 5BC所示, 与IgG1对照抗体相比, c7D3能促进PBMC分泌细胞因子IL-2和IFN-γ, 并且和抗PD-L1抗体联合用药效果优于两者各自单独用药效果, 表明c7D3能解除TIGIT对T细胞活化的抑制, 促进T细胞激活, 而且c7D3与抗PD-L1抗体可能具有协同的促T细胞激活作用。
以PD-(L)1免疫检查点抑制剂为代表的免疫疗法在多种不同肿瘤中具有良好的治疗效果, 但由于其他免疫检查点的参与或肿瘤本身的异质性, 临床上只有部分患者获益[18]。TIGIT是一种免疫检查点蛋白, 其在效应T细胞、NK细胞和Treg细胞中表达, 通过多种机制抑制T细胞和NK细胞的功能, 导致NK细胞耗竭[19, 20]。作为肿瘤治疗的新一代免疫检查点, TIGIT和其他检查点共同参与肿瘤的免疫逃逸, 靶向TIGIT单抗或与PD-(L)1抑制剂联合用药成为新的治疗方向[21-23]
目前全球尚无抗TIGIT抗体获批上市, 但已有十余款靶向TIGIT新药项目推进到临床试验阶段[24, 25]。在临床研究中, 抗TIGIT单抗多与抗PD-(L)1抗体以联合治疗方案用于各类实体瘤及血液系统肿瘤。罗氏制药的tiragolumab联合抗PD-L1抗体在转移性非小细胞肺癌及其他各类实体瘤的组合疗法中展现出良好的潜力, 被FDA认定为“突破性疗法”[26]; 默克公司的vibostolimab为人鼠嵌合单克隆抗体, 与pembrolizumab的联合治疗在晚期非小细胞肺癌患者中显示出良好的抗肿瘤活性和耐受性[27]; 百济神州生物科技有限公司的ociperlimab联合替雷利珠单抗治疗晚期非小细胞肺癌已处于Ⅲ期临床试验[28]。现有临床结果表明, 抗TIGIT单抗联合PD-(L)1抑制剂是一种很有前景的肿瘤联合免疫治疗方法。
本研究以人TIGIT胞外区蛋白为免疫原免疫小鼠, 通过常规细胞融合成功制备了一种新的抗TIGIT抗体c7D3。初步探讨c7D3生物学活性, 表明c7D3与TIGIT有较高亲和力及优良的阻断活性, 且细胞水平上具有良好生物功能活性, 证实抗体c7D3具有进一步开发成为肿瘤免疫治疗的候选分子的潜力。在下一步研究中, 本课题组将对c7D3分子进行人源化改造, 对药效学、成药性等方面分析优化, 进一步明确其成药可能, 为抗TIGIT抗体开发及肿瘤免疫治疗提供新的药物途径, 为更好的肿瘤治疗效果提供新的联合用药方案。
作者贡献: 吴振华和李娜为本文共同第一作者。吴振华完成了论文的修改, 设计并指导了所有实验; 李娜完成了论文撰写及抗体的生物学活性评价; 陈娟完成了杂交瘤细胞上清的检测及细胞株构建; 蒋美珠、陈瑶、梅小芬完成了结合和竞争方法开发; 王海彬完成了论文的审阅。
利益冲突: 所有作者声明均不存在利益冲突。
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2022年第57卷第11期
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doi: 10.16438/j.0513-4870.2022-0121
  • 接收时间:2022-01-26
  • 首发时间:2025-12-24
  • 出版时间:2022-11-12
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  • 收稿日期:2022-01-26
  • 修回日期:2022-03-25
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    浙江博锐生物制药有限公司, 浙江 台州 318000

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*王海彬, Tel: 86-576-89007929, E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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