A production process for lentivirus of chimeric antigen receptor targeting EpCAM that can meet industrial needs
[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 2.16×108 TU/mL,and a total amount of 2.16×1010 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.
epithelial cell adhesion molecule(EpCAM)chimeric antigen receptor(CAR)CAR-T therapylentivirus produce
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维普
