Chimeric antigen receptor T (CAR-T) cell therapy has the potential to transform cancer treatment. However, CAR-T therapy application is currently limited to certain types of relapse and refractory liquid tumors. To unlock the full potential of CAR-T therapy, technologic breakthroughs will be needed in multiple areas, including optimization of autologous CAR-T development, shortening the innovation cycle, and further manufacturing advancement of next-generation CAR-T therapies. Here, we established a simple and robust virus-free multiplex Quantum CART (qCART™) system that seamlessly and synergistically integrates four platforms: 1. GTailor™ for rapid identification of lead CAR construct design, 2. Quantum Nufect™ for effective but gentle electroporation-based gene delivery, 3. Quantum pBac™, featuring a virus-free transposon-based vector with large payload capacity and potentially safe integration profile, and 4. iCellar™ for robust and high-quality CAR+ T memory stem cell (TSCM) expansion. This robust, virus-free multiplex qCART™ system is expected to unleash the full potential of CAR-T therapy for treating diseases.Significance StatementChimeric antigen receptor T (CAR-T) cell therapy is currently ineffective against solid tumors. Here, we showcase Quantum CART (qCART™), a simple and robust system that seamlessly and synergistically integrates four platforms for optimal production of multiplex virus-free CAR-T cells: GTailor™ for rapid identification of lead CAR candidates; Quantum Nufect™ for effective but gentle electroporation-based gene delivery; Quantum pBac™, featuring a highly efficient, high payload capacity virus-free gene therapy vector and potentially safe integration profile; and iCellar™ for robust and high quality CAR-T cell expansion. We demonstrate that qCART™ is a simple and robust system for cost-effective and time-efficient manufacturing of T memory stem cell (TSCM) multiplex CAR-T cells.

Recent advances in gene therapy have brought novel treatment options to multiple fields of medicine, including cancer. However, safety concerns and limited payload capacity in commonly-utilized viral vectors prevent researchers from unlocking the full potential of gene therapy. Virus-free DNA transposons, including piggyBac, have been shown to obviate these shortcomings. We have previously demonstrated the superior transposition efficiency of a modified piggyBac system in HEK293 cells. Here, we further advanced and broadened the therapeutic application of this modified piggyBac system. We demonstrated that the internal domain sequence (IDS) within the 3′ terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. We showed that a plasmid-free donor vector having IDS-free terminal inverted repeats in conjunction with a helper plasmid expressing Quantum pBase™ v2 form the most optimal piggyBac system, Quantum pBac™ (qPB), in T cells. We further demonstrated that T cells transfected with qPB expressing CD20/CD19 CAR outperformed cells transfected with the same donor vector but with plasmid expressing hyPB transposase in CAR-T cell production. Importantly, we showed that qPB produced mainly CD8+ CAR-T cells that are also highly represented by TSCM. These CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells in vitro and in Raji-bearing immunodeficient mice. Our findings confirm that qPB is a promising virus-free vector system that is safer, and highly efficient in mediating transgene integration with the payload capacity to incorporate multiple genes.

CD19-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. Using a highly efficient piggyBac transposon-based vector, Quantum pBac, we developed a virus-free cell engineering system, Quantum CAR-T (qCART™), for development and production of multiplex CAR-T therapies. Here, we demonstrated in vitro and in vivo that consistent, robust, and functional CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using the qCART™ 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.

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. The GFP-based method demonstrated higher sensitivity in detecting CAR-T cell-mediated cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions.

Recent advances in gene therapy have brought novel treatment options for cancer. However, the full potential of this approach has yet to be unlocked due to the limited payload capacity of commonly utilized viral vectors. Virus‐free DNA transposons, including piggyBac, have the potential to obviate these shortcomings. In this study, we improved a previously modified piggyBac system with superior transposition efficiency. We demonstrated that the internal domain sequences (IDS) within the 3′ terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. Plasmid‐free donor vector devoid of IDS was used in conjunction with a helper plasmid expressing Quantum PBase™ v2 to generate an optimal piggyBac system, Quantum pBac™ (qPB), for use in T cells. qPB outperformed hyPB in CD20/CD19 CAR‐T production in terms of performance as well as yield of the CAR‐T cells produced. Furthermore, qPB also produced CAR‐T cells with lower donor‐associated variabilities compared to lentiviral vector. Importantly, qPB yielded mainly CD8+ CAR‐TSCM cells, and the qPB‐produced CAR‐T cells effectively eliminated CD20/CD19‐expressing tumor cells both in vitro and in vivo. Our findings confirm qPB as a promising virus‐free vector system with an enhanced payload capacity to incorporate multiple genes. This highly efficient and potentially safe system will be expected to further advance gene therapy applications.