靶向抗TIGIT和PVRIG双特异抗体的制备及体外生物性活性的研究  

Preparation and bioactivity of anti-TIGIT and PVRIG antibodies in vitro

作　　者：叶银松 米梓毓 雷毅 康金森[1,2] 郝锋 宁金鹰 杨建 YE Yinsong;MI Ziyu;LEI Yi;KANG Jinsen;HAO Feng;NING Jinying;YANG Jian(College of Pharmacy,Xinjiang Medical University,Urumqi 830017,China;Key Laboratory of Active Components of Xinjiang Natural Medicine and Drug Release Technology,Urumqi 830000,China;KYinno Biotechnology Co.,LTD,Beijing 100010,China)

机构地区：[1]新疆医科大学药学院,乌鲁木齐830017 [2]新疆天然药物活性组分与释药技术重点实验室,乌鲁木齐830000 [3]康源博创生物科技(北京)有限公司,北京100010

出　　处：《新疆医科大学学报》2024年第8期1153-1160,共8页Journal of Xinjiang Medical University

基　　金：新疆维吾尔自治区重大科技专项子课题(2022A03018-4);新疆医科大学博士科研启动基金(20210830)。

摘　　要：目的制备靶向抗T细胞免疫球蛋白-ITIM结构域蛋白(T cell immune receptor with Ig and ITIM domains,TIGIT)和脊髓灰质炎病毒受体相关免疫球蛋白结构域蛋白(Poliovirus receptor-related immunoglobin domain-containing protein,PVRIG)双特异抗体,探究其体外生物学活性。方法用PVRIG人源化抗体hP1和TIGIT人源化抗体hT1构建KY-TIGIT-hlgG4M-PVRIG-SCFV双特异性抗体,将纯化得到KY-TIGIT-hlgG4M-PVRIG-SCFV抗体进行SDS-PAGE凝胶电泳、热稳定性检测和稳定性分析;采用流式细胞术(Fluorescence activated cell sorting,FACS)检测双特异性抗体的结合活性;采用生物干涉膜技术(Bio-layer interferometry,BLI)检测双特异性抗体亲和力;用Jurkat-NFAT-Luc2-PD1-TIGIT-PVRIG细胞和293T-OS8-PVRL2-PDL1细胞中检测双特异性抗体KY-TIGIT-hlgG4M-PVRIG-SCFV对于TIGIT/PVRIG靶点的阻断作用;采用CMV recall assay法检测IFN-r在上清液中的浓度。结果SDS-PAGE凝胶电泳结果显示KY-TIGIT-hlgG4M-PVRIG-SCFV双特异性抗体纯度>95%;Tm结果为Tm1:66.34,Tm2:80.95。采用SEC-HPLC对双特异性抗体KY-TIGIT-hlgG4M-PVRIG-SCFV进行稳定性分析,其纯度均>90%。KY-TIGIT-hlgG4M-PVRIG-SCFV抗体和PVRIG的人源化抗体hP1与293T-PVRIG cell line的EC 50分别为0.16870μg/mL和0.03865μg/mL,与293T-cyno-PVRIG cell line的EC 50分别为0.03714μg/mL和0.03198μg/mL,与Human PVRIG Protein,His Tag的亲和力为1.74E-10M和1.69E-10M。KY-TIGIT-hlgG4M-PVRIG-SCFV抗体和TIGIT的人源化抗体hT1与293T-PVRIG cell line的EC 50分别为0.07027μg/mL和0.03121μg/mL;与293T-cyno-TIGIT cell line的EC 50分别为0.02028μg/mL和0.02822μg/mL;与Human TIGIT Protein,His Tag的亲和力为8.50E-10M和8.47E-10M。KY-TIGIT-hlgG4M-PVRIG-SCFV抗体具有阻断PD1/PDL1/TIGIT/PVRIG相互作用活性,且KY-TIGIT-hlgG4M-PVRIG-SCFV的EC 50为0.007μg/mL,优于单独人源化抗体hT1(EC 50为0.013μg/mL)和hP1(EC 50为0.048μg/mL)的作用。在含pp65 peptide条件下KY-TIGIT-hlgG4M-PVRIG-SCFV抗体分泌IFN-r为349.72 pg/mL,明显高于Objective:Preparation of targeted antibodies against T cell immune receptor with Ig and ITIM domains(TIGIT)and poliovirus receptor-related immunoglobin domain-containing protein(PVRIG)[HJ26x]and biological activity in vitro.Methods:KY-TIGIT-hlgG4M-PVRIG-SCFV bispecific antibodies were constructed with PVRIG humanized antibodies hP1 and TIGIT humanized antibody hT1,and the purification was raised to KY-TIGIT-hlgG4M-PVRIG-SCFV antibody for SDS-PAGE gel electrophoresis,thermal stability detection,and stability analysis;The binding activity of the bispecific antibody was determined by FACS;The binding activity of the bispecific antibody affinity was measured by BLI;The blocking effect of the bispecific antibody KY-TIGIT-hlgG4M-PVRIG-SCFV on TIGIT/PVRIG targets was tested in Jurkat-NFAT-Luc 2-PD1-TIGIT-PVRIG cells and 293T-OS8-PVRL 2-PDL 1 cells;The concentration of IFN-r in the supernatant was determined by the CMV recall assay assay.Results:SDS-PAGE gel electrophoresis results showed the KY-TIGIT-hlgG4M-PV RIG-SCFV bispecific antibody purity>95%.The Tm results were Tm1:66.34 and Tm2:80.95;Stability analysis by SEC-HPLC was KY-TIGIT-hlgG4M-PVRIG-SCFV,and the purity was>90%.KY-TIGIT-hlgG4M-PVRIG-SCFV antibody and humanized antibody hP1 for PVRIG had an EC 50 with 293T-PVRIG cell line of 0.16870μg/mL and 0.03865μg/mL,respectively.The EC 50 associated with the 293T-cyno-PVRIG cell line was 0.03714μg/mL and 0.03198μg/mL,respectively.The affinity with the Human PVRIG ProteinandHis Tag was 1.74E-10M and 1.69E-10M,respectively.KY-TIGIT-hlgG4M-PVRIG-SCFV antibody and humanized antibody hT1 for TIGIT had an EC 50 with 293T-TIGIT cell line of 0.07027μg/mL and 0.03121μg/mL,respectively.The EC 50 associated with the 293T-cyno-PVRIG cell line was 0.02028μg/m and 0.02822μg/mL,respectively.The affinity with Human PVRIG Protein and His Tag was 8.50E-10M and 8.47E-10M,respectively.KY-TIGIT-hlgG4M-PVRIG-SCFV antibody blocked PD1/PDL 1/TIGIT/PVRIG interaction activity and KY-TIGIT-hlgG4M-PVRIG-SCFV EC 50 of 0.007μg/mL was bett

关 键 词：T细胞免疫球蛋白和ITIM结构域蛋白 脊髓灰质炎病毒受体相关免疫球蛋白结构域蛋白 人源化抗体 双特异性抗体 生物学活性 

分 类 号：R392[医药卫生—免疫学]