GENERATION OF VIRUS-SPECIFIC T CELLS EXPRESSING A CD19 CAR (VST.CD19 CAR-T) FOR TREATMENT OF VIRAL INFECTIONS AND B CELL NEOPLASIA RELAPSE POST HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT)
- 1Instituto Nacional do Câncer (INCA), Rio de Janeiro, RJ, Brazil
Abstract
Introduction: Viral infections and disease relapse post allogeneic hematopoietic stem cell transplantation (HSCT) remain a significant burden, accounting for the majority of deaths after the procedure. Antiviral drug options are limited and highly toxic. Adoptive T cell therapy to reconstitute T cell immunity has proved safe and effective in HSCT recipients, but post-transplant relapse carries a dim prognosis. Patients with relapsed /refractory B cell malignancies can be rescued by immunotherapy with T-cells genetically modified with chimeric antigen receptors (CARs) for CD19 (CD19.CARTs). However, CAR-T therapy access is limited in developing countries due to high costs and technology availability. Objectives: Integration of virus-specific T cell (VST) and CAR-T platforms would address two of the most relevant problems after allogenic HSCT, and by designing an inexpensive manufacturing protocol, we planned to reduce cost. We used a non-viral platform (Sleeping Beauty - SB transposon system) to generate VST.CD19 CAR-T cells which preserve the functionality of both receptors, TCR and CAR, and direct cytotoxic activity against tumor cells and virus-infected cells. Materials and methods: The protocol consists of magnetic selection of the memory fraction of T cells (CD45RA negative) from peripheral blood mononuclear cells (PBMCs) from healthy donors; activation with anti-CD3/CD28 for 48 hours followed by electroporation of isomolar fractions of minicircles, the first containing the gene of interest (CAR anti-CD19) and the second containing the transposase (SB). Minicircles are minimal expression cassettes generated by removing bacterial genes from parental plasmids, thereby significantly reducing cellular toxicity. Following electroporation, cells are activated with irradiated autologous PBMCs pulsed with viral pepmixes (CMV, EBV, BKV, and ADV) and expanded in vitro. Results: On day 11, the percentage of cells expressing CAR ranged from 12-47% (n = 7 donors). Viability remained above 90%, with cells expanded 6 to 10-fold. IFN-γ ELISPOT analysis showed preservation of viral specificity, especially for CMV and EBV. The immunophenotype profile consists mostly of effector T cells (Teff and Temra), with low expression of exhaustion markers. In vitro lysis assays against CD19+ cell lines showed cytotoxic activity at effector:target ratios up to 1:2. VST.CD19 CAR-T single cell RNA sequencing analysis is in progress. Discussion and Conclusion: The results confirm the proposed protocol allows for generation of VST.CD19 CAR-T cells using a non-viral platform with a transposon system, and minicircles as DNA vectors. This platform is GMP-compliant and compatible with the manufacture of clinical products. VST.CD19 CAR-T cells have the potential to address two of the main complications of hematopoietic stem cell transplantation (HSCT), and the cost reduction may increase patient access to new technologies within the Brazilian public health system (SUS).
Related
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BibTeX
@article{mazzi2026generation,
title = {{GENERATION OF VIRUS-SPECIFIC T CELLS EXPRESSING A CD19 CAR (VST.CD19 CAR-T) FOR TREATMENT OF VIRAL INFECTIONS AND B CELL NEOPLASIA RELAPSE POST HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT)}},
author = {Mariana Torres MAZZI and Paula Daniela Souza ALVES and Martín Hernán BONAMINO},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.106873},
url = {https://doi.org/10.1016/j.htct.2026.106873},
note = {CC BY},
}
RIS
TY - JOUR
TI - GENERATION OF VIRUS-SPECIFIC T CELLS EXPRESSING A CD19 CAR (VST.CD19 CAR-T) FOR TREATMENT OF VIRAL INFECTIONS AND B CELL NEOPLASIA RELAPSE POST HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT)
AU - Mariana Torres MAZZI
AU - Paula Daniela Souza ALVES
AU - Martín Hernán BONAMINO
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.106873
UR - https://doi.org/10.1016/j.htct.2026.106873
AB - Introduction: Viral infections and disease relapse post allogeneic hematopoietic stem cell transplantation (HSCT) remain a significant burden, accounting for the majority of deaths after the procedure. Antiviral drug options are limited and highly toxic. Adoptive T cell therapy to reconstitute T cell immunity has proved safe and effective in HSCT recipients, but post-transplant relapse carries a dim prognosis. Patients with relapsed /refractory B cell malignancies can be rescued by immunotherapy with T-cells genetically modified with chimeric antigen receptors (CARs) for CD19 (CD19.CARTs). However, CAR-T therapy access is limited in developing countries due to high costs and technology availability. Objectives: Integration of virus-specific T cell (VST) and CAR-T platforms would address two of the most relevant problems after allogenic HSCT, and by designing an inexpensive manufacturing protocol, we planned to reduce cost. We used a non-viral platform (Sleeping Beauty - SB transposon system) to generate VST.CD19 CAR-T cells which preserve the functionality of both receptors, TCR and CAR, and direct cytotoxic activity against tumor cells and virus-infected cells. Materials and methods: The protocol consists of magnetic selection of the memory fraction of T cells (CD45RA negative) from peripheral blood mononuclear cells (PBMCs) from healthy donors; activation with anti-CD3/CD28 for 48 hours followed by electroporation of isomolar fractions of minicircles, the first containing the gene of interest (CAR anti-CD19) and the second containing the transposase (SB). Minicircles are minimal expression cassettes generated by removing bacterial genes from parental plasmids, thereby significantly reducing cellular toxicity. Following electroporation, cells are activated with irradiated autologous PBMCs pulsed with viral pepmixes (CMV, EBV, BKV, and ADV) and expanded in vitro. Results: On day 11, the percentage of cells expressing CAR ranged from 12-47% (n = 7 donors). Viability remained above 90%, with cells expanded 6 to 10-fold. IFN-γ ELISPOT analysis showed preservation of viral specificity, especially for CMV and EBV. The immunophenotype profile consists mostly of effector T cells (Teff and Temra), with low expression of exhaustion markers. In vitro lysis assays against CD19+ cell lines showed cytotoxic activity at effector:target ratios up to 1:2. VST.CD19 CAR-T single cell RNA sequencing analysis is in progress. Discussion and Conclusion: The results confirm the proposed protocol allows for generation of VST.CD19 CAR-T cells using a non-viral platform with a transposon system, and minicircles as DNA vectors. This platform is GMP-compliant and compatible with the manufacture of clinical products. VST.CD19 CAR-T cells have the potential to address two of the main complications of hematopoietic stem cell transplantation (HSCT), and the cost reduction may increase patient access to new technologies within the Brazilian public health system (SUS).
SN - 2531-1379
SN - 2531-1387
ER -