首页|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

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

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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 re-ceptor-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 sor-ting,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 EC50 分别为 0.168 70 μg/mL 和 0.038 65 µg/mL,与 293T-cyno-PVRIG cell line 的 EC50 分别为 0.037 14 μg/mL 和 0.031 98 µg/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 EC50 分别为 0.070 27 μg/mL 和 0.031 21 µg/mL;与293T-cyno-TIGIT cell line 的 EC50 分别为 0.020 28μg/mL 和 0.028 22 μg/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 EC50为 0.007 μg/mL,优于单独人源化抗体 hT1(EC50为 0.013 μg/mL)和 hP1(EC50为 0.048 μg/mL)的作用.在含 pp65 peptide 条件下 KY-TIGIT-h1gG4M-PVRIG-SCFV 抗体分泌 IFN-r 为 349.72 pg/mL,明显高于其他抗体.结论 KY-TIGIT-hlgG4M-PVRIG-SCFV抗体具有高亲和力和良好的生物学活性,有望开发成为肿瘤免疫治疗的抗体药物.
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)and biological activity in vitro.Methods KY-TIGIT-hlgG4M-PVRIG-SCFV bispecific antibodies were con-structed 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 sta-bility 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 EC50 with 293T-PVRIG cell line of 0.168 70 μg/mL and 0.038 65 μg/mL,respec-tively.The EC50 associated with the 293T-cyno-PVRIG cell line was 0.037 14 μg/mL and 0.031 98 μg/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 EC50 with 293T-TIGIT cell line of 0.070 27 μg/mL and 0.031 21 μg/mL,respectively.The EC50 associat-ed with the 293T-cyno-PVRIG cell line was 0.020 28 μg/m and 0.028 22 μg/mL,respectively.The affini-ty 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 EC50 of 0.007 μg/mL was better than humanized antibody hT1(EC50=0.013 μg/mL)and hP1(EC50=0.048 μg/mL).IFN-r secretion of 349.72 pg/mL by KY-TIGIT-hlgG4M-PVRIG-SCFV antibody was significantly higher than the other antibodies.Conclusion KY-TIGIT-hlgG4M-PVRIG-SCFV antibodies,with high affinity and good biological activity,are expected to be developed as new anti-body drugs for tumor immunotherapy.

T cell immune receptor with Ig and ITIM domains(TIGIT)poliovirus receptor-related immu-noglobin domain-containing protein(PVRIG)humanized antibodiesdouble specific antibodybioactivity

叶银松、米梓毓、雷毅、康金森、郝锋、宁金鹰、杨建

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新疆医科大学药学院,乌鲁木齐 830017

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

康源博创生物科技(北京)有限公司,北京 100010

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

新疆维吾尔自治区科技重大专项子课题新疆医科大学博士科研启动基金

2022A03018-420210830

2024

10.3969/j.issn.1009-5551.2024.08.018

新疆医科大学学报
新疆医科大学

新疆医科大学学报

CSTPCD
影响因子：0.76
ISSN：1009-5551
年,卷(期)：2024.47(8)