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
BACKGROUND: The efficiency of chimeric antigen receptor (CAR) T-cell-based therapies depends on a sufficient expansion of CAR T cells in vivo and can be weakened by intra-tumoral suppression of CAR T cell functions, leading to a failure of therapy. For example, certain B-cell malignancies such as chronic lymphocytic leukemia are weakly sensitive to treatment with CAR T cells. Co-expression of proinflamatory cytokines such as IL-12 and IL-18 by CAR T cells have been shown to enhance their antitumor function. We similarly engineered CAR T cell to co-express IL-21 and studied the effects of IL-21 on CAR T cells specific to CD19 and prostate-specific membrane antigens using an in vitro co-culture model and NSG mice transplanted with B-cell tumors. RESULTS: IL-21 enhanced the expansion of CAR T cells after antigenic stimulation, reduced the level of apoptosis of CAR T cells during co-culture with tumor cells and prevented differentiation of CAR T cells toward late memory phenotypes. In addition, induced secretion of IL-21 by CAR T cells promoted tumor infiltration by CD19-specific CAR (CAR19) T cells in NSG mice, resulting in reduced tumor growth. By co-culturing CAR19 T cells with bone-marrow fragments infiltrated with CLL cells we demonstrate that IL-21 reduces the immunosupressive activity of CLL cells against CAR19 T cells. CONCLUSIONS: CAR19 T cells armed with IL-21 exhibited enhanced antitumor functions. IL-21 promoted their proliferation and cytotoxicity against chronic lymphocytic leukemia (CLL). The results suggest that arming CAR T cells with IL-21 could boost the effectiveness of CAR T-mediated therapies.
- Klíčová slova
- CAR T cells, chronic lymphocytic leukemia, interleukin 21, piggyBac transposon,
- MeSH
- fenotyp MeSH
- imunosupresivní léčba MeSH
- interleukiny metabolismus MeSH
- lidé MeSH
- myši MeSH
- nádory imunologie terapie MeSH
- proliferace buněk MeSH
- receptory antigenů T-buněk metabolismus MeSH
- T-lymfocyty imunologie MeSH
- transpozibilní elementy DNA genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CD19-specific chimeric antigen receptor MeSH Prohlížeč
- Interleukin-21 MeSH
- interleukiny MeSH
- receptory antigenů T-buněk MeSH
- transpozibilní elementy DNA MeSH
BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. RESULTS: We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. CONCLUSIONS: Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.
- Klíčová slova
- CAR T cells, IL-21, IL-4, IL-7, cancer immunotherapy, piggyBac transposon,
- MeSH
- aktivace lymfocytů účinky léků genetika MeSH
- buněčné kultury metody MeSH
- buňky PC-3 MeSH
- chimerické antigenní receptory genetika metabolismus MeSH
- elektroporace MeSH
- genetické vektory MeSH
- HEK293 buňky MeSH
- imunoterapie adoptivní metody MeSH
- interleukin-4 farmakologie MeSH
- interleukin-7 farmakologie MeSH
- interleukiny farmakologie MeSH
- kultivované buňky MeSH
- Lentivirus genetika MeSH
- lidé MeSH
- proteinové inženýrství metody MeSH
- protinádorové vakcíny genetika imunologie MeSH
- receptory antigenů T-buněk genetika metabolismus MeSH
- T-lymfocyty * cytologie účinky léků imunologie metabolismus MeSH
- transdukce genetická metody MeSH
- transpozibilní elementy DNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chimerické antigenní receptory MeSH
- Interleukin-21 MeSH
- interleukin-4 MeSH
- interleukin-7 MeSH
- interleukiny MeSH
- protinádorové vakcíny MeSH
- receptory antigenů T-buněk MeSH
- transpozibilní elementy DNA MeSH
Our work examined the production of intracellular interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-4 by in vitro stimulated CD3+ cells from 38 chronic myeloid leukemia (CML) patients. At the time of diagnosis the percentages of cells producing INF-γ, TNF-α, and IL-2 were strongly suppressed compared to those in healthy control subjects. Hematological remission achieved through treatment with tyrosine-kinase inhibitors was associated with a highly significant increase in the ratio of cells producing all 4 cytokines. The percentages of CD3+ cells producing cytokines were dependent on age, more so in CML patients than in healthy controls, and they negatively correlated with the number of leukocytes. Patients with an optimal therapy outcome possessed higher percentages of cytokine-producing CD3+ cells at diagnosis than those with nonoptimal outcomes. This difference was statistically significant in the case of INF-γ-producing cells, and it was on the brink of significance in the case of IL-2-producing cells.
- Klíčová slova
- Chronic myeloid leukemia, Interferon-γ, Interleukin-2, Interleukin-4, Intracellular cytokine production, Tumor necrosis factor-α,
- MeSH
- antigeny CD3 metabolismus MeSH
- chronická myeloidní leukemie farmakoterapie imunologie MeSH
- cytokiny biosyntéza MeSH
- dospělí MeSH
- interferon gama biosyntéza MeSH
- interleukin-2 biosyntéza MeSH
- interleukin-4 biosyntéza MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- T-lymfocyty imunologie MeSH
- techniky in vitro MeSH
- TNF-alfa biosyntéza MeSH
- tumor burden imunologie MeSH
- věkové faktory MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny CD3 MeSH
- cytokiny MeSH
- IL2 protein, human MeSH Prohlížeč
- IL4 protein, human MeSH Prohlížeč
- interferon gama MeSH
- interleukin-2 MeSH
- interleukin-4 MeSH
- TNF protein, human MeSH Prohlížeč
- TNF-alfa MeSH
Serum samples taken at diagnosis in 28 chronic myeloid leukemia patients were tested for the presence of 20 cytokines by a magnetic bead-based Bio-plex immunoassay. According to complete cytogenetic remission achieved at 12 months of treatment, patients were divided into groups with either optimal or non-optimal outcome. Patients with increased cytokine levels tended to react optimally to the therapy more frequently than those others. TGF-β3 was a notable exception; its levels were significantly higher in patients with non-optimal outcomes. Further analysis enabled us to define two combinations of cytokine cut-off levels - namely low TGF-β3 and either high IL-8 or high MCP-1-each of which corresponded to therapy outcome better than either Sokal or EUTOS scores.
- Klíčová slova
- Chronic myeloid leukemia, EUTOS, Prediction reliability, Serum cytokines, Sokal,
- MeSH
- biologické markery MeSH
- chronická myeloidní leukemie krev diagnóza mortalita terapie MeSH
- cytokiny krev MeSH
- dospělí MeSH
- Kaplanův-Meierův odhad MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- prediktivní hodnota testů MeSH
- prognóza MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
- cytokiny MeSH
Myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha, formally known as CDC42BPA) is a serine/threonine kinase that can regulate actin/myosin assembly and activity. Recently, it has been shown that it possesses a functional iron responsive element (IRE) in the 3'-untranslated region (UTR) of its mRNA, suggesting that it may be involved in iron metabolism. Here we report that MRCKalpha protein expression is also regulated by iron levels; MRCKalpha colocalizes with transferrin (Tf)-loaded transferrin receptors (TfR), and attenuation of MRCKalpha expression by a short hairpin RNA silencing construct leads to a significant decrease in Tf-mediated iron uptake. Our results thus indicate that MRCKalpha takes part in Tf-iron uptake, probably via regulation of Tf-TfR endocytosis/endosome trafficking that is dependent on the cellular cytoskeleton. Regulation of the MRCKalpha activity by intracellular iron levels could thus represent another molecular feedback mechanism cells could use to finely tune iron uptake to actual needs.
- MeSH
- DM-kinasa MeSH
- endocytóza MeSH
- endozomy enzymologie MeSH
- HeLa buňky MeSH
- lidé MeSH
- malá interferující RNA genetika MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- receptory transferinu metabolismus MeSH
- RNA interference MeSH
- transferin metabolismus MeSH
- železo metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CDC42BPA protein, human MeSH Prohlížeč
- DM-kinasa MeSH
- malá interferující RNA MeSH
- protein-serin-threoninkinasy MeSH
- receptory transferinu MeSH
- transferin MeSH
- železo MeSH
