Optimized GMP-grade production of non-viral Sleeping Beauty-generated CARCIK cells for enhanced fitness and clinical scalability

Abstract

Background: Strict adherence to GMP guidelines and regulatory compliance is crucial when transitioning from research to clinical-grade production of ATMPs like CAR T cells. The success of CAR T cell therapy in treating hematological malignancies highlights the need for closed or automated systems to ensure quality and efficacy. Recent evidence also suggests that ex vivo culture conditions can significantly impact CAR T cell functionality.

Methods: We present our optimized methodology for expanding Sleeping Beauty transposon-engineered Chimeric Antigen Receptor-Cytokine-Induced Killer (CARCIK) cells using G-Rex devices and evaluate its impact on CARCIK cell phenotype and T cell fitness.

Results: Building on our previously validated protocol, we introduced key simplifications to optimize the CARCIK differentiation process. Delaying the nucleofection step eliminated the need for feeder cells while maintaining efficient CAR expression and high cell viability. Transitioning from T-flasks to G-Rex bioreactors reduced operator hands-on time from 21 to 28 days to 14-17 days and resulted in a less differentiated CARCIK cell product. Metabolic and transcriptional analyses showed that the novel protocol improves CARCIK cell fitness and in vivo efficacy against B-cell lymphoma. The novel method was validated in Good Manufacturing Practices (GMP) conditions at our two Cell Factories and yielded enough numbers of CARCIK-CD19 cells for clinical use.

Conclusions: Optimizing non-viral CARCIK cell production using G-Rex bioreactors and refined timing adjustments has streamlined the workflow, enhanced cell fitness, and resulted in a highly effective therapeutic product with demonstrated in vivo efficacy in mice. These improvements reduced manipulation and contamination risks, while optimizing logistics and space efficiency, facilitating allogeneic CARCIK generation for a current phase I/II clinical trial (NCT05869279) in patients with R/R CD19 + non-Hodgkin Lymphoma (B-cell NHL) and Chronic Lymphocytic Leukemia (CLL), confirming the approach's scalability and clinical potential.

Keywords: CAR T cells; Good manufacturing practices (GMP); Hematological malignancies; Sleeping beauty transposon; T cell fitness; Workflow optimization.

Fig. 1

Optimization of non-viral CARCIK cell platform. (A–C) CARCIK-CD19 cells were generated with titrated doses of pt4-CD19CAR and SB100X plasmids (in ug: 10 + 0.5; 10 + 1; 7.5 + 0.5; 7.5 + 1) and expanded in G-Rex or Flask. (A) Total cell yields at the end of the cell culture (starting from 10 × 10⁶ cells); (B) Percentage of CAR expression at the end of the differentiation gated on the CD3⁺ subset; (C) Percentage of CD3⁺CD56⁺ cells at the end of the differentiation. (D–G) CARCIK-CD19 cells were produced using either the standard Day 0 protocol (Protocol 1) or the feeder-free Day 2 protocol and expanded in G-Rex or Flasks (Protocol 2). (D) Total cell yields at the end of the cell culture (starting from 10 × 10⁶ cells); (E) Percentage of CAR expression; (F) Percentage of CD3⁺CD56⁺ cells; (G) Percentage of CD4⁺ and CD8⁺ cells obtained between two different methods both in Flasks and G-Rex. (H) Short-term in vitro cytotoxicity of CARCIK-CD19 cells produced using the new Day 2 feeder-free protocol (in T-flasks and G-Rex) against the CD19⁺ REH cell line, tested at an E: T ratio of 1:5. Results represent four independent experiments. (I) Vector copy number analysis (plasmid/cell) and transposase (copies/10000 GUS) gene expression by RT-PCR at the end of culture for CARCIK-CD19 cells produced with the Day 2 feeder-free protocol and expanded in G-Rex. (L) Schematic of the standard protocol in Flasks (Protocol 1) and optimized feeder free method in G-Rex (Protocol 2)

Fig. 2

Differentiation and metabolic profiles of CARCIK cells. (A) Immunophenotype along with CAR expression of CARCIK-CD19 cells produced following Protocol 2. (B) Memory phenotype comparison between Protocol 1- and Protocol 2- derived CARCIK-CD19 cells. (C–E) Intradonor memory and metabolic differences (analyzed by Seahorse T Cell Metabolic Kit) between Flask- and G-Rex-derived CARCIK-CD19 cells generated following Protocol 2. OCR: Oxygen Consumption Rate ECAR: Extracellular Acidification Rate

Fig. 3

Gene expression characterization of Flask- and G-Rex derived CARCIK-CD19 cells. (A) Hierarchical clustering heatmap of Flask- and G-Rex- derived CARCIK-CD19 cells produced following Protocol 2. (B) Volcano plot of the differential gene expression analysis of Flask- and G-Rex- derived CARCIK-CD19. (C–E) Pathway analysis and (F) number of DEGs significantly up- and downregulated comparing Flask- and G-Rex- derived CARCIK-CD19 cells

Fig. 4

Dose finding study of G-Rex derived CARCIK-CD19 cells (Protocol 2) in a survival DAUDI mouse model. (A) DAUDI-engrafted NSG mice model scheme. Mice were sub-lethally irradiated (2 Gy) and 0.5 × 10⁶ DAUDI cells were injected i.v. on Day 0. Mice were treated with 5 × 10⁶ (5 M), 10 × 10⁶ (10 M), and 15 × 10⁶ (15 M), CARCIK-CD19 cells at Day2 and bled every 10 days to quantify disease burden. (B) Analysis of hCD45⁺CD19⁺ cells/ml in PB of untreated (DAUDI only) and treated (5 M, 10 M, and 15 M CARCIK-CD19 cells) mice. (C) Kaplan Meier survival curves of NSG mice engrafted with DAUDI cells, untreated or treated with CARCIK-CD19 cells. Comparisons of survival curves were determined by Log-rank test. (D) Scheme of DAUDI-FFLuc + engrafted NSG mice. Mice were sub-lethally irradiated (2 Gy) and 0.5 × 10⁶ DAUDI-FFLuc + cells were injected i.v. on Day0. Mice were treated with 5 × 10⁶ (5 M), 10 × 10⁶ (10 M), and 15 × 10⁶ (15 M), CARCIK-CD19 cells at Day2 and bioluminescence was performed weekly to follow the disease burden. (E) Tumor burden of DAUDI FFLuc + cells in untreated and treated mice measured by bioluminescent imaging from day 20 after DAUDI injection. (F) Kaplan Meier survival curves of DAUDI engrafted NSG mice, left untreated or treated with CARCIK‐CD19 cells. Comparisons of survival curves were determined by Log-rank test. (G) BLI imaging was performed using the IVIS lumina III imaging system. Tumor burden was visualized on days 20, 29, 36, 41, 50 and 57. (H) RNAscope in situ hybridization images showing CARCIK-CD19 and DAUDI cells in the spleen of untreated and treated (10 M and 15 M of CARCIK-CD19 cells) mice. Upper panels Representative pseudo-color fluorescence images showing automated staining for CD3 (green) on FFPE mouse spleen sections. The samples represent mice injected with DAUDI cells alone (left), DAUDI + CARCIK-CD19 cells (10 M, center), or DAUDI + CARCIK-CD19 cells (15 M, right). Representative Duplex RNAscope images of different FFPE slides of the same samples using probes targeting CAR (Channel 1, green dots) and CD19 (Channel 2, red dots). Images are shown at 40x magnification. Scale bars: 60 μm