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

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3' and 5' arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 108 CAR19 T cells per one electroporation of 1 × 107 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products.

• Klíčová slova
• CD19, T cells, chimeric antigenic receptor, electroporation, piggyBac transposon,
• Publikační typ
• časopisecké články MeSH

Chimeric antigen receptor T-cells (CAR T-cells) represent a novel and promising approach in cancer immunotherapy. According to the World Health Organization (WHO), the number of oncological patients is steadily growing in developed countries despite immense progress in oncological treatments, and the prognosis of individual patients is still relatively poor. Exceptional results have been recorded for CAR T-cell therapy in patients suffering from B-cell malignancies. This success opens up the possibility of using the same approach for other types of cancers. To date, the most common method for CAR T-cell generation is the use of viral vectors. However, dealing with virus-derived vectors brings possible obstacles in the CAR T-cell manufacturing process owing to strict regulations and high cost demands. Alternative approaches may facilitate further development and the transfer of the method to clinical practice. The most promising substitutes for virus-derived vectors are transposon-derived vectors, most commonly sleeping beauty, which offer great coding capability and a safe integration profile while maintaining a relatively low production cost. This review is aimed at summarizing the state of the art of nonviral approaches in CAR T-cell generation, with a unique perspective on the conditions in clinical applications and current Good Manufacturing Practice. If CAR T-cell therapy is to be routinely used in medical practice, the manufacturing cost and complexity need to be as low as possible, and transposon-based vectors seem to meet these criteria better than viral-based vectors.

• MeSH
• buněčné kultury metody   MeSH
• chimerické antigenní receptory genetika   imunologie   MeSH
• genetické vektory genetika   MeSH
• imunoterapie adoptivní metody   MeSH
• lidé MeSH
• nádory imunologie   terapie   MeSH
• T-lymfocyty imunologie   transplantace   MeSH
• technika přenosu genů * MeSH
• transpozibilní elementy DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• chimerické antigenní receptory MeSH
• transpozibilní elementy DNA MeSH