Clinical-scale Production of Anti-cd19 Car-t Cells Using a Non-viral System Based on Minicircle (mc) Plasmids and the Sleeping Beauty (sb) Transposon
Year: 2026
Type: article
Abstract: Background CD19-directed chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable clinical outcomes in patients with B-cell malignancies. However, current manufacturing protocols rely predominantly on viral vectors, making production costly, technically complex, and difficult to scale. We implemented a non-viral platform based on minicircle (MC) plasmids and the Sleeping Beauty (SB) transposon system to generate anti-CD19 CAR-T cells. MCs are compact DNA vectors derived from parental plasmids in genetically engineered bacteria expressing an inducible recombinase. MCs retain only the CAR transgene under a specific promoter while eliminating the prokaryotic backbone, thereby improving safety and vector compactness for gene therapy applications. Objective To establish a scalable manufacturing protocol for anti-CD19 CAR-T cells using the Sleeping Beauty transposon system. Methods T cells isolated from leukapheresis products or leukocyte reduction filters obtained from healthy donors were purified using anti-CD3 magnetic beads and activated with anti-CD3/CD28 beads for 48 h. Cells were nucleofected (MaxCyte GTx) with the PT4-19BBz vector and SB100X transposase, followed by expansion in the G-Rex culture system (Wilson Wolf) supplemented with interleukin-2. CAR expression, viability, and cell expansion were monitored over 12 days. Results Different MC doses, cell seeding densities, and electroporation programs optimized for activated T cells were evaluated. By day 6 (D6), all conditions maintained viability ≥ 85%, with CAR expression ranging from 16.1% to 73.1%, while D12 values ranged from 13.6% to 47.0%. As expected, donor-dependent variability was observed, with fold expansion ranging from 0.5–4.0 at D6 and 1.6–13.4 at D12. Nevertheless, all conditions achieved a target number of CAR-T cell yield for manufacturing by D6. After establishing the protocol at small scale (OC-100 × 2), the process was successfully scaled to low/medium clinical scale (CL-1.1) and large scale (R20K). Comparable performance was obtained across all three manufacturing platforms, with 29.7±5.0% CAR+ cells at D6 and 22.7±3.6% at D12, viability of 93–99% from D6 onward, fold expansion of 1.2–4.0 at D6 and 6.2–13.4 at D12, and a 0.3–1.3-fold (D6) and 1.5–3.3-fold (D12) increase in CAR-T cell number relative to the pre-electroporation T-cell input. Discussion The SB/MC platform achieved clinically relevant CAR expression without viral vectors, while G-Rex culture consistently supported expansion and preserved high viability across donors, starting materials, and manufacturing scales. Increasing MC dose improved gene transfer efficiency without compromising viability. Although the percentage of CAR-positive cells decreased after D6, continued cell proliferation resulted in a sustained increase in the absolute number of CAR-T cells through D12. Importantly, the target CAR-T cell yield was consistently achieved by D6 regardless of MC dose, donor, starting material, or production scale. Conclusion This non-viral manufacturing platform consistently generated anti-CD19 CAR-T cells with high viability and stable CAR expression. These findings support shortened manufacturing timelines and reduced production costs and complexity compared with viral vector-based approaches, strengthening the translational potential of this strategy.
Institutions: Instituto Nacional de Câncer - INCA, Fundação Oswaldo Cruz
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: diamond
OpenAlex ID: W7223140951