一种可满足产业化需求的靶向EpCAM嵌合抗原受体慢病毒生产工艺

A production process for lentivirus of chimeric antigen receptor targeting EpCAM that can meet industrial needs

目的建立一种可满足产业化需求的靶向上皮细胞粘附分子(EpCAM)嵌合抗原受体(CAR)慢病毒生产工艺。方法以EpCAM蛋白为抗原,免疫小鼠获得EpCAM单抗,采用EpCAM单抗的scFv为胞外单链可变区,构建人源化靶向EpCAM的第三代CAR载体质粒,并通过EpCAM CAR载体质粒与慢病毒包装质粒共转染HEK 293T细胞获得慢病毒粗毒液,粗毒液经核酸酶孵育、过滤澄清、Core 700层析、浓缩换液、除菌过滤、制剂分装等工序获得EpCAM慢病毒成品。结果通过EpCAM单抗成功构建了靶向EpCAM抗原的嵌合抗原受体Humanized EpCAM ScFv-CD28-CD3ζ-CAR,并通过该EpCAM CAR进行2 L悬浮体系慢病毒包装所收获粗毒液,经层析及超滤浓缩等纯化工艺收获慢病毒成品100 mL,成品慢病毒转导滴度达2.16×10^(8)TU/mL,慢病毒总量达2.16×10^(10)TU。成功开发了可满足产业化需求的靶向EpCAM CAR慢病毒上游包装及下游纯化生产工艺。结论具有成本效益的慢病毒(LV)载体制备对于满足产业化需求至关重要,本研究在EpCAM蛋白、EpCAM抗体、及EpCAM CAR载体质粒的基础上,结合完整的慢病毒悬浮生产工艺,制备高滴度高纯度的靶向EpCAM嵌合抗原受体慢病毒,为EpCAM CAR-T细胞治疗实体瘤应用及其产业化奠定基础。

【Objective】To establish a production process for targeted epithelial cell adhesion molecule(EpCAM)chimeric antigen receptor lentivirus that can meet the needs of industrialization.【Methods】The EpCAM protein was used as the antigen to immunize mice and obtain EpCAM monoclonal antibodies.The scFv of the EpCAM monoclonal antibody was used as the extracellular single chain variable region to construct a humanized third-generation chimeric antigen receptor(EpCAM CAR)vector plasmid targeting EpCAM.The crude lentiviral solution was obtained by co-transfecting the EpCAM CAR vector plasmid with the lentiviral packaging plasmid into HEK 293T cells.The crude solution was incubated with nuclease,filtered and clarified,subjected to Core 700 chromatography,concentrated and changed,and sterilized and filtered obtaining EpCAM lentivirus finished products through processes such as formulation packaging.【Results】A chimeric antigen receptor Humanized was targeting EpCAM antigen was successfully constructed using EpCAM monoclonal antibody,and the crude venom was obtained from 2 L suspension system lentivirus packaging using this EpCAM CAR.After purification processes such as chromatography and ultrafiltration concentration,100 mL of the finished lentivirus product was obtained,with a lentivirus transduction titer of 2.16×10^(8) TU/mL,and a total amount of 2.16×10^(10) TU.A targeted EpCAM CAR lentivirus upstream packaging and downstream purification production process that can meet industrial needs was successfully developed.【Conclusion】The preparation of cost-effective lentivirus(LV)vectors is crucial for meeting industrial needs.This study,based on EpCAM protein,EpCAM antibody,and EpCAM CAR vector plasmid,combined with a complete lentivirus suspension production process,prepared a high titer and high purity targeted EpCAM chimeric antigen receptor lentivirus,laying the foundation for the application and industrialization of EpCAM CAR-T cell therapy for solid tumors.