Quantum CART(qCART), apiggyBac-basedsystem for development and production of virus-free multiplex CAR-T cell therapy

Abstract

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.

Manufacturing CD20/CD19-targeted iCasp9 regulatable CAR-TSCMcells usingqCART, theQuantum pBac-based CAR-T system

“…Genotoxicity is also a predominant safety issue associated with gene therapy products. Consistent with our previous study, we observed low risk of genotoxicity in qCART TM -manufactured CAR-T cells, given the low integrant copy number, safe integration profile, low enhancer activity, and low residual Quantum pBase, suggesting minimal integrant remobilization risks 45 (Supplementary Figure 1B; Figure 2B). Furthermore, acute toxicities in patients in response to CAR-T therapies, such as CRS and ICANS, have been major concerns in CAR-T development.…”

“…4,19,20 While we also used aAPC in our initial experiments, we could generate sufficient quantity of patient-derived qCART™ -modified CAR-T cells without aAPC-induced enrichment. Our recent study further confirmed that qCART™ -manufactured CAR-T cells may be more effective in tumor control without aAPC enrichment 45 . Here, we demonstrated that 2.29×10 8 –1.38×10 9 T cells could be generated in a 1 L-G-Rex within a short time-frame of 12 days using T cells from patients with B-cell malignancies.…”

“…Consistent with our previous study, we observed low risk of genotoxicity in qCART™-manufactured CAR-T cells, given the low integrant copy number, safe integration profile, low enhancer activity, and low residual qPBase, suggesting minimal integrant remobilization risks (Supplementary Figure 1B; Figure 2B). 45 Acute toxicities in patients in response to CAR-T therapies, such as CRS and ICANS, have been major concerns in CAR-T development. [18][19][20] Furthermore, CAR-T cells have the potential to transform into cancer cells; a rare case of CAR + CD4 T cell lymphoma has been reported recently in patients treated with CD19 CAR-T cells.…”

“…14 We recently developed the most advanced version of piggyBac known as Quantum pBac TM (qPB) that is 15 times more active and potentially safer than hyperactive piggyBac in human T cells 44,45 . In our previous studies, we successfully developed a novel genetic engineering system known as Quantum CAR-T (qCART TM ) that combines efficient transgene design, evaluation, and identification of lead construct (GTailor TM ); virusfree vector (Quantum pBac TM ); effective gene delivery (Quantum Nufect TM ); and optimized cell expansion (iCellar TM ) 44,45 . We reported that qCART TM could be generically used to engineer highly potent CAR-T stem cell memory (T SCM ), a T cell population associated with enhanced anti-tumor efficacy in cancer patients.…”

Quantum pBac: An effective, high-capacitypiggyBac-based gene integration vector system for unlocking gene therapy potential

“…Given the low frequency of footprint-induced mutation by piggyBac (< 5 %) and the fact that only a minimal portion (< 0.4 %) of CAR + T cells expressed detectable level of qPBase in CAR-T cells (Figure 5B), transposase in a plasmid form should be sufficiently safe when applied to highly proliferative ex vivo -engineered cells. Additionally, our recent genome-wide integration profiling of qPB in CAR-T products derived from two distinct donors further support its safe use in T cell engineering(52).…”