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

Abstract

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.

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

“…Of note, we observed that increasing the amount of donor DNA (carrying CAR) introduced into CAR-T cells increases integrant copy number in cells. 26 To reduce safety risks, we optimized the concentration of donor DNA such that integrant copy number per cell would be below five, as shown by the low CAR copy numbers found in T cells of both donors (3.08 and 1.68 in donors 1 and 2, respectively; Supplementary Table S2). Interestingly, remarkable conformity was found in the integration profiles of both donor T cells.…”

“…One possibility is that qBT increased the CD4 CAR + T SCM population (Figure 4C), which in turn enhanced proliferation of CD8 T SCM population without concomitant differentiation. This may reflect the high (3-9) and balanced (approximately 1) 26 CD8:CD4 ratios observed in healthy donors and patients, respectively.…”

“…The CD19/CD20 tandem CAR-T with iCasp9 is an exemplary product which possesses all of these desired features. 26 Possible mechanisms that contribute to the desired high CAR-T SCM , balanced CD8:CD4 ratio, and reduced safety concerns are discussed below.…”

“…[17][18][19][20] Our studies have demonstrated that almost all qCART™-generated CAR-T cell products contain >90% TN and TSCM (> 70% TSCM in most cases) in both CD4 and CD8 CAR + T cell populations, regardless of (1) CAR construct design, (2) GOI size, and (3) PBMC source (healthy donors or patients with B-cell malignancies; Figure 4, Table 2). 26 We believe this desired feature is synergistically achieved by the combined benefits derived from: (1) including 4-1BB rather than CD28 in the CAR construct, (2) using qNF instead of HTCN for nucleofection, (3) choosing qPB over virus-based or other virus-free (e.g. hyPB) vector systems, and (4) using iCellar™, qBT in particular, for CAR-T cell expansion.…”

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

“…As demonstrated in another study, this preferential enrichment of CD8 T cells can serve to complement the more difficult-to-enrich patient CD8 T cells and contribute to a balanced CD8/CD4 ratio (approximately 1.0) in CAR-T cells produced from patients with B-cell malignancies. 44 Additionally, CAR-T cell expansion and persistence have emerged as key efficacy determinants in cancer patients, and both are positively correlated with the proportion of T SCM cells in the final CAR-T cell product 50,51 . In line with these findings, we have demonstrated that our qPB-derived CAR-T cells, which are enriched in TSCM cells (especially among CD8 + CAR + T cells), exhibited potent antitumor effects both in vitro and in vivo.…”