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
- 1Instituto Nacional de Câncer (INCA), Rio de Janeiro, RJ, Brazil
- 2Instituto Nacional de Câncer (INCA), Rio de Janeiro, RJ, Brazil; Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ, Brazil
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.
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BibTeX
@article{santana2026clinicalscale,
title = {{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}},
author = {Lívia Pimentel de SANT'ANA and Arthur Da Silva NEUMANN and Emmanuel Arthur Albuquerque ARAGÃO and Luiza De Macedo ABDO and Paula Daniela Souza ALVES and Sabrina Alves dos REIS and Martín Hernán BONAMINO},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.106839},
url = {https://doi.org/10.1016/j.htct.2026.106839},
note = {CC BY},
}
RIS
TY - JOUR
TI - 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
AU - Lívia Pimentel de SANT'ANA
AU - Arthur Da Silva NEUMANN
AU - Emmanuel Arthur Albuquerque ARAGÃO
AU - Luiza De Macedo ABDO
AU - Paula Daniela Souza ALVES
AU - Sabrina Alves dos REIS
AU - Martín Hernán BONAMINO
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.106839
UR - https://doi.org/10.1016/j.htct.2026.106839
AB - 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.
SN - 2531-1379
SN - 2531-1387
ER -