Manufacturing CD20/CD19-targeted iCasp9 regulatable CAR-TSCM cells using a Quantum pBac-based CAR-T engineering system

Abstract

CD19-targeted chimeric antigen receptor (CAR) T cell therapies have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CD19-CAR-T-treated patients experience 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 numbers of virally engineered CAR-T cells. Using a highly efficient piggyBac transposon-based vector, Quantum pBac™ (qPB), we developed a virus-free cell-engineering system for development and production of multiplex CAR-T therapies. Here, we demonstrate in vitro and in vivo that consistent, robust and functional CD20/CD19 dual-targeted CAR-T stem cell memory (CAR-TSCM) cells can be efficiently produced for clinical application using qPB™. In particular, we showed that qPB™-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. Therefore, the simplicity of manufacturing multiplex CAR-T cells using the qPB™ system has the potential to improve efficacy and broaden the accessibility of CAR-T therapies.

Fig 1. Effect of qBT and aAPC on CAR-T cells.

(A) Schematic diagram of the CAR20/19 CAR construct. (B-E) Human peripheral blood mononuclear cells (PBMC) electroporated with Quantum pBac™-expressing CAR and cultured for 10 days in the presence or absence of aAPC and/or Quantum Booster™ (qBT) were harvested and assessed for (B) cell expansion fold change, (C) percentage CAR⁺ of live cells (CD3⁺ PI^-, >90%), and percentage of T_SCM cell subsets in (D) CD4⁺ and (E) CD8⁺ cells. Data shown are from six healthy donors. Horizontal lines represent the mean and s.e.m. fold change (B), mean and s.e.m. percentage of CAR⁺ cells (C), and mean and s.e.m. percentage of T_SCM cell subsets (D and E). * p < 0.05, ** p < 0.01, *** p < 0.001. (F) Schematic diagram of the CARiC9-20/19 CAR construct. A T2A sequence enables co-expression of iCasp9 and CD20/CD19-targeted scFvs under the EF1α promoter. (G-I) In vivo functional characterization of CAR-T cells produced by perfusion culture system. (G) In vivo cytotoxicity of CAR-T cells with or without pre-incubation with aAPC in Raji-GFP/Luc-bearing immunodeficient mice. Fluorescence intensity values (H) and survival curves (I) of mice from (G) plotted against time. Results shown are from 4 to 8 mice/group. T cells were obtained from a representative donor. Vehicle and Pan-T cells (non-engineered T cells) were used as controls. qBT was present in all cell culture conditions. Groups were compared by log-rank (Mantel-Cox) test, *p < 0.05, **p < 0.01.

Fig 2. Characteristics of CARiC9-20/19 CAR-T cells.

Percentages of (A) CAR⁺ and (B) transposase⁺ (qPBase⁺) cells were analyzed by flow cytometry on days 1, 8 and 10 after nucleofection. (C) Fold change of CARiC9-20/19 CAR-T cells after 10 days of culture in G-Rex are shown. (D) Percentage of CD4 and CD8 T cells in the CAR⁺ population on day 10 after nucleofection. (E) Distributions of T_N, T_SCM, T_CM, T_EM, and T_EFF subsets in the CD4 (upper panel) and CD8 populations (lower panel). (F) Expression of exhaustion markers PD-1, TIM-3, and LAG-3 in CARiC9-20/19 CAR-T cells at 10 days post-nucleofection. (G) Expression of senescence markers KLRG-1 and CD57 in CARiC9-20/19 CAR-T cells. (A-G) Data represent mean ± SD for 9 healthy donors, n = 9. Each set of histogram plots shown in (C), (D), (E), and (F) represents one donor.

Fig 3. In vitro functional analysis of CARiC9-20/19 CAR-T cells.

(A-B) CAR-T cells derived from two healthy donors were assessed for cytotoxicity against Raji-GFP/Luc cells by Celigo image cytometry. Groups were compared by One-way ANOVA with Tukey multiple comparison, ***p < 0.001, **p < 0.01, *p < 0.05. (C) IFN-γ, (D) TNF-α, and (E) IL-2 secretion by CARiC9-20/19 CAR-T cells following antigen stimulation was determined by ELISA. Pan-T cells (non-modified cells) served as a control group. Data represent mean ± SD, n = 3. Groups were compared by Student’s t-test, ***p < 0.001. NS, not statistically significant.

Fig 4. Anti-tumor activity of CARiC9-20/19 CAR-T cells in a B-cell lymphoma immunodeficient xenograft mouse model.

(A) Schematic diagram of in vivo Raji xenograft model experimental design. (B-C) Bioluminescence imaging was performed to monitor tumor cell persistence (n = 4/group) and tumor growth were quantified by total flux (photon/s). (D) Kaplan-Meier survival curves for the different treatment groups are shown. **p < 0.01, *p < 0.05. NS, not statistically significant.

Fig 5. Anti-tumor activity of CARN87 cells in a gastric cancer immunodeficient xenograft mouse model.

(A) Schematic diagram of the CARN87 CAR construct. (B) Performance of CAR-T cells produced using lentiviral and non-viral qPB cell production. (C) Schematic diagram of in vivo NCI-N87 xenograft model experimental design. (D) Bioluminescent imaging was performed to monitor tumor cell persistence (n = 3/group). (E) Human T cell counts of blood samples taken from the indicated group of mice at day 10 and day 37 post-CAR-T injection.

Fig 6. Assessment of cancer-patient-derived CARiC9-20/19 CAR-T cells.

T cells derived from healthy donors and cancer patients were nucleofected with CARiC9-20/19. (A) CD8/CD4 ratios were assessed before (PBMC) and after (CAR-T) nucleofection. (B) Distribution of T_N, T_SCM, T_CM, T_EM, and T_EFF subsets in CD4⁺ (upper panel) and CD8⁺ (lower panel) CAR-T cell populations among the patient samples in (A). DLBCL, diffuse large B-cell lymphoma; CLL, chronic lymphocytic leukemia; HL, Hodgkin’s lymphoma; MM, multiple myeloma.