Manufacturing CD20/CD19-targeted iCasp9 regulatable CAR-TSCMcells usingqCART, theQuantum pBac-based CAR-T system
Authors: Chang Peter S. , Chen Yi-Chun , Hua Wei-Kai , Hsu Jeff C. , Tsai Jui-Cheng , Huang Yi-Wun , Kao Yi-Hsin , Wu Pei-Hua , Chang Yi-Fang , Chang Ming-Chih , Chang Yu-Cheng , Jian Shiou-Ling , Lai Jiann-Shiun , Lai Ming-Tain , Yang Wei-Cheng , Shen Chia-Ning , Wen Kuo-Lan Karen , Wu Sareina Chiung-Yuan 
Published: May 2022

ABSTRACTBackgroundCD19-targeted chimeric antigen receptor therapies (CAR19) have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CAR19-treated patients experienced progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore, it remains a challenge to efficiently, cost-effectively, and consistently manufacture clinically relevant number of virally engineered CAR-T cells.MethodsUsing a highly efficientpiggyBactransposon-based vector,Quantum pBac, we developed a virus-free cell engineering system,Quantum CART (qCART™), for development and production of multiplex CAR-T therapies.ResultsHere, we demonstratedin vitro and in vivothat consistent, robust, and functional CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using theqCART™ system for clinical application.qCART™-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug.ConclusionsTheqCART™ system is an elegant system for the manufacturing of CAR-T products having all the desired CAR-T therapy attributes. We believe that the simplicity of manufacturing multiplex CAR-T cells using theqCART™ system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future.What is already known on this topicDespite the considerable success which has been achieved with CD19-targeted chimeric antigen receptor therapies (CAR19), >50% of CAR19-treated patients still experienced progressive disease. Therefore, there is a need to further improve CAR19 therapies. Current CAR19 therapies commonly utilize virus-based cell engineering methods. CAR-T production using these methods face multiple hurdles, including difficulties to efficiently, cost-effectively, and consistently manufacture clinically relevant number of CAR-T cells. We have previously used a highly efficientpiggyBactransposon-based vector,Quantum pBac, to establishQuantum CART(qCART™) which is a virus-free cell engineering system for development and production of multiplex CAR-T therapies.What this study addsIn this report, we further demonstratein vitroandin vivothat consistent, robust, and functional iCasp9-regulatable, CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using theqCART™ system for clinical application. These cells possess all the desired attributes for ensuring therapeutic efficacy in CAR-T therapy, including high CAR-TSCM, balanced CD8/CD4 ratio, low exhaustion and senescence marker expressions, and highex vivoandin vivoexpansion capacity. Importantly, we show thatqCART™-manufactured CAR-T cells from hematological cancer patients expanded efficiently, effectively eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. We believe that the simplicity of manufacturing multiplex CAR-T cells using theqCART™ system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future.How this study might affect research, practice or policyOur findings demonstrate thatqCART™ is a virus-free CAR-T engineering system for manufacturing CAR-TSCMcells from either healthy donors or hematological cancer patients, that possess all the desired attributes for a successful CAR-T therapy. These cells expanded efficiently, rapidly eradicated tumors, and can be safely controlled via activation of iCasp9. We expect that this simple yet robust system for manufacturing multiplex CAR-T cells will advance the CAR-T field.