Cells were cultured in OpTmizer medium (Thermo Fisher) supplemented with Quantum Booster (GenomeFrontier Therapeutics) in G-Rex 24- or 6M-well plates (Wilson Wolf) or conventional plates for 10 days. Irradiated artificial antigen presenting cells (aAPC), CD19-expressing K562 cells, were added to electroporated T cells at 1:1 ratio on day 3. An iCasp9-CD20/CD19-CAR transposon minicircle carrying CD20 scFv and CD19 scFv and a qPBase plasmid were delivered into activated T cells using 4D-Nucleofector device (Lonza) and Quantum Nufect Kit (GenomeFrontier Therapeutics) according to the manufacturer’s instructions. Primary human CD3 + T cells were isolated from PBMCs by negative selection using EasySep Human T Cell Isolation Kit (StemCell Technologies) or by positive selection using Dynabeads Human T-Expander CD3/CD28 (Thermo Fisher), followed by activation of isolated T cells with Dynabeads (3:1 bead-to-cell ratio) for two days. PBMCs were extracted using Ficoll-Hypaque gradient separation (Cytiva). We demonstrate enhanced anti-tumor efficacy of GF-CART01 cells both in vitro and in a xenotransplant immunodeficient mouse model.įrozen peripheral blood mononuclear cells (PBMCs) from adult healthy donors and patients were obtained from Chang Gung Memorial Hospital (IRB approval #201900578A3) and MacKay Memorial Hospital (IRB approval #20MMHIS330e), respectively. Here, we further validate that the qCART™ system can be used to rapidly manufacture highly potent patient-derived bicistronic CD20/CD19-targeted iCasp9 regulatable CAR-T cells (GF-CART01) for treating B-cell malignancies. We reported that qCART™ could be used to engineer highly potent T stem cell memory (T SCM) CAR-Tcells. We previously developed a novel genetic engineering system known as Quantum CART (qCART™ ) that combines efficient transgene design, evaluation, and identification of lead construct ( GTailor) virus-free vector ( Quantum pBac) effective gene delivery ( Quantum Nufect) and optimized cell expansion ( iCellar). We recently developed the most advanced version of piggyBac known as Quantum pBac (qPB) that is 15 times more active than hyperactive piggyBac in human T cells. ![]() Transposon system, such as Sleeping Beauty and piggyBac, is a virus-free alternative for generating CAR-T cells but suffers from low gene transfer efficiency and limited expansion of engineered cells. As demonstrated by recent clinical studies, bispecific CD19/CD20-targeted CAR-T therapy improved patient survival compared to CAR19 therapy. Evidence suggest that downregulation of CD19 may in part be responsible for patient relapse. However, treatment with CAR19 therapy resulted in only 30-40% long-term progression-free survival (PFS) in aggressive Non-Hodgkin lymphoma (NHL) patients, and the median event-free survival (EFS) of adult B-cell ALL patients was 6.1 months. (2003) first demonstrated the eradication of B-cell tumors by CD19-targeted second-generation CAR-T cells, several commercial CAR-T products have been approved by FDA. To date, B-cell malignancies remain the most actively-studied cancers for CAR-T therapy development. Furthermore, virus-based gene therapies have limited gene payload capacity, making them less suitable for engineering multiplex armored CAR-T cells. Frequent cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) associated with clinical patients treated with conventional CAR-T therapies have raised safety concerns. ![]() Over the last decade, chimeric antigen receptor (CAR)-T therapy has become one of the most promising cancer treatment, but the heavy reliance on lentiviral and retroviral vectors for CAR-T cell manufacturing makes the therapy expensive and inaccessible to the masses. We believe that the simplicity of manufacturing multiplex CAR-T cells using the qCART™ 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. Importantly, we show that qCART™-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. These cells possess all the desired attributes for ensuring therapeutic efficacy in CAR-T therapy, including high CAR-T SCM, balanced CD8/CD4 ratio, low exhaustion and senescence marker expressions, and high ex vivo and in vivo expansion capacity. What this study adds In this report, we further demonstrate in vitro and in vivo that consistent, robust, and functional iCasp9-regulatable, CD20/CD19 dual-targeted CAR-T stem cell memory (T SCM) cells can be efficiently manufactured using the qCART™ system for clinical application.
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