BACKGROUND: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. METHODS: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. RESULTS: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. CONCLUSIONS: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.

• Klíčová slova
• CAR-T cells, PiggyBac PB transposon, electroporation, leukemia, lymphoma,
• MeSH
• akutní myeloidní leukemie * terapie   imunologie   genetika   MeSH
• allogeneické buňky imunologie   MeSH
• antigeny CD19 * imunologie   genetika   MeSH
• B-buněčný lymfom terapie   imunologie   genetika   MeSH
• chimerické antigenní receptory * genetika   imunologie   MeSH
• elektroporace MeSH
• imunoterapie adoptivní * metody   MeSH
• lidé MeSH
• podkladové buňky MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• transpozibilní elementy DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD19 * MeSH
• chimerické antigenní receptory * MeSH
• transpozibilní elementy DNA * MeSH

T-cell malignancies can be divided into precursor (T-acute lymphoblastic leukemia/lymphoblastic lymphoma, T-ALL/LBL) and mature T-cell neoplasms, which are comprised of 28 different entities. Most of these malignancies are aggressive with rather poor prognosis. Prognosis of relapsed/refractory (R/R) disease is especially dismal, with an expected survival only several months after progression. Targeted therapies, such as antiCD30 immunotoxin brentuximab vedotin, antiCD38 antibody daratumumab, and anti-CCR4 antibody mogamulizumab are effective only in subsets of patients with T-cell neoplasms. T-cells equipped with chimeric antigen receptor (CAR-Ts) are routinely used for treatment of R/R B-cell malignancies, however, there are specific obstacles for their use in T-cell leukemias and lymphomas which are fratricide killing, risk of transfection of malignant cells, and T-cell aplasia. The solution for these problems relies on target antigen selection, CRISPR/Cas9 or TALEN gene editing, posttranslational regulation of CAR-T surface antigen expression, and safety switches. Structural chromosomal changes and global changes in gene expression were observed with gene-edited products. We identified 49 studies of CAR-based therapies registered on www.clinicaltrials.gov. Most of them target CD30 or CD7 antigen. Results are available only for a minority of these studies. In general, clinical responses are above 50% but reported follow-up is very short. Specific toxicities of CAR-based therapies, namely cytokine release syndrome (CRS), seem to be connected with the antigen of interest and source of cells for manufacturing. CRS is more frequent in antiCD7 CAR-T cells than in antiCD30 cells, but it is mild in most patients. More severe CRS was observed after gene-edited allogeneic CAR-T cells. Immune effector cell associated neurotoxicity (ICANS) was mild and infrequent. Graft-versus-host disease (GvHD) after allogeneic CAR-T cells from previous hematopoietic stem cell donor was also observed. Most frequent toxicities, similarly to antiCD19 CAR-T cells, are cytopenias. CAR-based cellular therapy seems feasible and effective for T-cell malignancies, however, the optimal design of CAR-based products is still unknown and long-term follow-up is needed for evaluation of their true potential.

• Klíčová slova
• CAR-T cells, T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma, T-cell lymphoma, chimeric antigen receptor (CAR), immunotherapy, therapy,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH