Simplified process for the production of anti–CD19-CAR–engineered T cells

Background aimsAdoptive immunotherapy with the use of chimeric antigen receptor (CAR)-engineered T cells specific for CD19 has shown promising results for the treatment of B-cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. We describe a new, simplified method to produce anti-CD19-CAR T cells.MethodsT cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 h, and a second transduction was then performed. No spinoculation was used. Cells were then expanded for an additional 9 days.ResultsThe method was validated through the use of two PBMC products from a patient with B-cell chronic lymphoblastic leukemia and one PBMC product from a healthy subject. The two PBMC products from the patient with B-cell chronic lymphoblastic leukemia contained 11.4% and 12.9% T cells. The manufacturing process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6-fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first four patients with acute lymphoblastic leukemia treated at our institution.ConclusionsWe developed a simplified, semi-closed system for the initial selection, activation, transduction and expansion of T cells with the use of anti-CD3/anti-CD28 beads and bags to produce autologous anti-CD19 CAR–transduced T cells to support an ongoing clinical trial.     

Optimization of lentiviral vector transduction into peripheral blood mononuclear cells in combination with the fibronectin fragment CH-296 stimulation

Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a na?ve phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the na?ve phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.     

Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use

Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 10⁶ CD3⁺ cells/cm² of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph⁺ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.     

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

Abstract Background aims. Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results. Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.     

Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering

Background aimsMost current chimeric antigen receptor (CAR) T cells are generated by viral transduction, which induces persistent expression of CARs and may cause serious undesirable effects. Messenger RNA (mRNA)-based approaches in manufacturing CAR T cells are being developed to overcome these challenges. However, the most common method of delivering mRNA to T cells is electroporation, which can be toxic to cells.MethodsThe authors designed and engineered an exosome delivery platform using the bacteriophage MS2 system in combination with the highly expressed protein lysosome-associated membrane protein 2 isoform B on exosomes.ResultsThe authors’ delivery platform achieved specific loading and delivery of mRNA into target cells and achieved expression of specific proteins, and anti-CD3/CD28 single-chain variable fragments (scFvs) expressed outside the exosomal membrane effectively activated primary T cells in a similar way to commercial magnetic beads.ConclusionsThe delivery of CAR mRNA and anti-CD3/CD28 scFvs via designed exosomes can be used for ex vivo production of CAR T cells with cancer cell killing capacity. The authors’ results indicate the potential applications of the engineered exosome delivery platform for direct conversion of primary T cells to CAR T cells while providing a novel strategy for producing CAR T cells in vivo.     

A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.     

Suspension culture on microcarriers and as aggregates enables expansion and differentiation of pluripotent stem cells (PSCs)

Background aimsHuman pluripotent stem cells (PSCs) hold a great promise for promoting regenerative medical therapies due to their ability to generate multiple mature cell types and for their high expansion potential. However, cell therapies require large numbers of cells to achieve desired therapeutic effects, and traditional two-dimensional static culture methods cannot meet the required production demand for cellular therapies. One solution to this problem is scaling up expansion of PSCs in bioreactors using culture strategies such as growing cells on microcarriers or as aggregates in suspension culture.MethodsIn this study, we directly compared PSC expansion and quality parameters in microcarrier- and aggregate-cultures grown in single-use vertical-wheel bioreactors.ResultsWe showed comparable expansion of cells on microcarriers and as aggregates by day 6 with a cell density reaching 2.2 × 10⁶ cells/mL and 1.8 × 10⁶ cells/mL and a fold-expansion of 22- and 18-fold, respectively. PSCs cultured on microcarriers and as aggregates were comparable with parallel two-dimensional cultures and with each other in terms of pluripotency marker expression and retention of other pluripotency characteristics as well as differentiation potential into three germ layers, neural precursor cells and cardiomyocytes.ConclusionsOur study did not demonstrate a clear advantage between the two three-dimensional methods for the quality parameters assessed. This analysis adds support to the use of bioreactor systems for large scale expansion of PSCs, demonstrating that the cells retain key characteristics of PSCs and differentiation potential in suspension culture.     

Demonstrating the Manufacture of Human CAR‐T Cells in an Automated Stirred‐Tank Bioreactor

Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 10⁶ cells ml‐1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T‐flasks and gas‐permeable culture bags). Importantly, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.     

Transition from serum-supplemented monolayer to serum-free suspension lentiviral vector production for generation of chimeric antigen receptor T cells

Background aimsLentiviral vectors (LVs) have been used extensively in gene therapy protocols because of their high biosafety profile and capacity to stably express a gene of interest. Production of these vectors for the generation of chimeric antigen receptor (CAR) T cells in academic and research centers is achieved using serum-supplemented static monolayer cultures. Although efficient for pre-clinical studies, this method has a number of limitations. The main hurdles are related to its incompatibility with robust and controlled large-scale production. For this reason, cell suspension culture in bioreactors is desirable. Here the authors report the transition of LV particle production from serum-supplemented monolayer to serum-free suspension culture with the objective of generating CAR T cells.MethodsA self-inactivating LV anti-CD19 CAR was produced by transient transfection using polyethylenimine (PEI) in human embryonic kidney 293 T cells previously adapted to serum-free suspension culture.ResultsLV production of 8 × 10⁶ transducing units (TUs)/mL was obtained in serum-supplemented monolayer culture. LV production in the serum-free suspension conditions was significantly decreased compared with monolayer production. Therefore, optimization of the transfection protocol was performed using design of experiments. The results indicated that the best condition involved the use of 1 μg of DNA/10⁶ cells, 1 × 10⁶ cells/mL and PEI:DNA ratio of 2.5:1. This condition used less DNA and PEI compared with the standard, thereby reducing production costs. This protocol was further improved with the addition of 5 mM of sodium butyrate and resulted in an increase in production, with an average of 1.5 × 10⁵ TUs/mL. LV particle functionality was also assessed, and the results indicated that in both conditions the LV was capable of inducing CAR expression and anti-tumor response in T cells, which in turn were able to identify and kill CD19+ cells in vitro.ConclusionsThis study demonstrates that the transition of LV production from small-scale monolayer culture to scalable and controllable bioreactors can be quite challenging and requires extensive work to obtain satisfactory production.     

Bispecific targeting of CD20 and CD19 increases polyfunctionality of chimeric antigen receptor T-cell products in B-cell malignancies

Background aimsSelective immune pressure contributes to relapse due to target antigen downregulation in patients treated with anti-CD19 chimeric antigen receptor (CAR) T cells. Bispecific lentiviral anti-CD20/anti-CD19 (LV20.19) CAR T cells may prevent progression/relapse due to antigen escape. Highly polyfunctional T cells within a CAR T-cell product have been associated with response in single-antigen-targeted anti-CD19 CAR T cells.MethodsThe authors performed a single-cell proteomic analysis to assess polyfunctional cells in our LV20.19 CAR T-cell product. Analysis was limited to those treated at a fixed dose of 2.5 × 10⁶ cells/kg (n = 16). Unused pre-infusion CAR T cells were thawed, sorted into CD4/CD8 subsets and stimulated with K562 cells transduced to express CD19 or CD20. Single-cell production of 32 individual analytes was measured and polyfunctionality and polyfunctional strength index (PSI) were calculated.ResultsFifteen patients had adequate leftover cells for analysis upon stimulation with CD19, and nine patients had adequate leftover cells for analysis upon stimulation with CD20. For LV20.19 CAR T cells, PSI was 866–1109 and polyfunctionality was 40–45%, which were higher than previously reported values for other CAR T-cell products.ConclusionsStimulation with either CD19 or CD20 antigens resulted in similar levels of analyte activation, suggesting that this product may have efficacy in CD19– patient populations.     

Novel xeno-free and serum-free culturing condition to improve piggyBac transposon-based CD19 chimeric antigen receptor T-cell production and characteristics

Background aimsChimeric antigen receptor (CAR) T cell is a novel therapy for relapse and refractory hematologic malignancy. Characteristics of CAR T cells are associated with clinical efficacy and toxicity. The type of serum supplements used during cultivation affects the immunophenotype and function of viral-based CAR T cells. This study explores the effect of serum supplements on nonviral piggyBac transposon CAR T-cell production.MethodsPiggyBac CD19 CAR T cells were expanded in cultured conditions containing fetal bovine serum, human AB serum or xeno-free serum replacement. We evaluated the effect of different serum supplements on cell expansion, transduction efficiency, immunophenotypes and antitumor activity.ResultsXeno-free serum replacement exhibited comparable CAR surface expression, cell expansion and short-term antitumor activity compared with conventional serum supplements. However, CAR T cells cultivated with xeno-free serum replacement exhibited an increased naïve/stem cell memory population and better T-cell expansion after long-term co-culture as well as during the tumor rechallenge assay.ConclusionsOur study supports the usage of xeno-free serum replacement as an alternative source of serum supplements for piggyBac-based CAR T-cell expansion.     

Retrovirus mediated gene transduction of human T-cell subsets

Purpose: Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukaemia. Graft versus host disease (GVHD) is a potentially life threatening complication of AlloBMT mediated by the T cells contained within the graft. In order to be able to control GVHD, the allogeneic T cells may be transduced with a suicide gene such as herpes simplex virus thymidine kinase (HSV-tk). For this strategy to be successful, all subsets of T cells should be transduced to a similar extent. Also, the transduction protocol should not induce expression of unwanted homing receptors, nor should it lead to unwanted skewing of the T-cell receptor repertoire. We have studied the transduction efficiency of naïve and memory subsets of CD4+ and CD8+ T cells, and examined the transduced T-cell subsets for possible changes in T-cell receptor (TCR) repertoire and homing receptor expression. Methods: The cells were transduced using a Moloney murine retroviral vector carrying a conjugate of the genes encoding the truncated form of the cell surface marker, low affinity nerve growth factor receptor (LNGFR) and HSV-tk. Transduction efficiency and homing receptor expression were quantified by flow cytometry. TCR repertoire was determined by spectratyping. Results: We obtained a transduction efficiency of 30–50% of the cells, with no difference between the T-cell subsets. Cell surface receptors responsible for homing to skin, gastrointestinal tract or lymph nodes were practically absent at the end of 2 weeks in culture. The activation procedure seemed to favour the expansion of certain T-cell clones over polyclonal populations. However, there was no difference in the TCR repertoire between transduced and non-transduced cells. Conclusion: Changes in the composition of the T-cell subsets at the end of the cell culture were the results of the activation, and not the suicide gene transduction. The transduced T cells did not express unwanted homing receptors.     

Clinical-grade,large-scale,feeder-free expansion of highly active human natural killer cells for adoptive immunotherapy using an automated bioreactor

Background aimsNatural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality.ResultsSignificant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 × 10⁹ NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks.ConclusionsThese results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.     

Selective depletion of FOXP3high cells by Fas–Fas-L–induced apoptosis occurs in CD4+CD25+-enriched populations during repeated expansion

Background aimsExpansion of anti-CD25 bead-isolated human Tregs culture has paradoxically resulted in reduced suppressive activity, but the mechanism(s) responsible for these observations are poorly defined.MethodsMagnetic-bead isolated human CD25⁺ cells were expanded with anti-CD3/CD28 beads and high doses of rhIL-2. Detection of Fas and Fas ligand (Fas-L) expression, activation of Caspase 8, cell proliferation and cytokine production was evaluated by multi-color fluorescence-activated cell sorting analysis. The role of Fas–Fas-L–mediated cell death was dissected through the use of agonist or antagonist monoclonal antibodies directed at Fas and Fas-L.ResultsRepeated expansion of bead-enriched CD4⁺CD25⁺ cells generated a cellular product with markedly reduced suppressive activity and with significantly increased CD8⁺ T cells and CD4⁺ T cells producing interferon-γ and/or interleukin-2. We showed that Fas–Fas-L–mediated apoptosis of CD4⁺FOXP3^high cells and rapid cell-cycling of CD8⁺ T cells were collectively responsible for the reduced proportion of CD4⁺FOXP3^high cells in expanded cultures. The depletion of CD4⁺FOXP3^high cells and activation of Caspase 8 in CD4⁺FOXP3^high cells was attenuated by Fas antagonist antibody, ZB4, in short-term culture. However, the loss of CD4⁺FOXP3^high cells during expansion was not prevented by either Fas or Fas-L antagonist antibodies.ConclusionsTaken together, the data show that Fas–Fas-L–mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.     

Genetically engineered fixed K562 cells: potent “off-the-shelf” antigen-presenting cells for generating virus-specific T cells

Background aimsThe human leukemia cell line K562 represents an attractive platform for creating artificial antigen-presenting cells (aAPC). It is readily expandable, does not express human leukocyte antigen (HLA) class I and II and can be stably transduced with various genes.MethodsIn order to generate cytomegalovirus (CMV) antigen-specific T cells for adoptive immunotherapy, we transduced K562 with HLA-A10201 in combination with co-stimulatory molecules.ResultsIn preliminary experiments, irradiated K562 expressing HLA-A10201 and 4-1BBL pulsed with CMV pp65 and IE-1 peptide libraries failed to elicit antigen-specific CD8⁺ T cells in HLA-A10201⁺ peripheral blood mononuclear cells (PBMC) or isolated T cells. Both wild-type K562 and aAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcal enterotoxin B (SEB) and OKT3 and in mixed lymphocyte reaction (MLR). Transwell experiments suggested that suppression was mediated by a soluble factor; however, MLR inhibition was not reversed using transforming growth factor-β blocking antibody or prostaglandin E₂ inhibitors. Full abrogation of the suppressive activity of K562 on MLR, SEB and OKT3 stimulation was only achieved by brief fixation with 0.1% formaldehyde. Fixed, pp65 and IE-1 peptide-loaded aAPC induced robust expansion of CMV-specific T cells.ConclusionsFixed gene-modified K562 can serve as effective aAPC to expand CMV-specific cytotoxic T lymphocytes for therapeutic use in patients after stem cell transplantation. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemia and other tumors.     

Transiently redirected T cells for adoptive transfer

Background aimsT cells can be redirected to reject cancer by retroviral transduction with a chimeric antigen receptor (CAR) or by administration of a bispecific T cell engager (BiTE). We demonstrate that transfection of T cells with messenger (m) RNA coding for CAR is an alternative strategy.MethodsWe describe the pre-clinical evaluation of a method based on transient modification of expanded T cells with a CD19 CAR directed against B-cell malignancies. CAR mRNA was generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation was used to load the mRNA into the cytoplasm of T cells with no risk of insertional mutagenesis.ResultsAfter transfection > 80% of T cells were viable, with 94% CAR expression. Transfected T cells were cytolytic to CD19⁺ targets and produced interferon (IFN)-γ in response. Killing of CD19⁺ target cells was demonstrated even at day 8 with undetectable CAR expression. Increasing the concentration of mRNA resulted in higher surface CAR expression, better killing and more IFN-γ release but at the expense of increased activation-induced cell death. Finally, we demonstrated that a second transgene could be introduced by co-electroporation of CXCR4 or CCR7 with CAR to also modify chemotactic responses.ConclusionsWe advocate the transient redirection approach as well suited to meet safety aspects for early phase studies, prior to trials using stably transduced cells once CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in pre-clinical studies.     

CD19 chimeric antigen receptor–redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide–derived dendritic cell vaccine in leukemia

BackgroundChimeric antigen receptor (CAR)–T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy.MethodsWe pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells.ResultsThe Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity.DiscussionThis study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.     

Consequences of adjusting cell density and feed frequency on serum-free expansion of thymic regulatory T cells

BackgroundGiven the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents.MethodsSeeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality.ResultsThe authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 10⁵ cells/cm²) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 10⁵ cells/cm²), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures.ConclusionThese data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype.     

Stem-like memory T cells are generated during hollow fiber perfusion-based expansion and enriched after cryopreservation in an automated modular cell therapy manufacturing process

Background aimsModular automation is a flexible and reliable option to build the foundation of a new or evolving process or to introduce automation to a process that is already established. Herein the authors demonstrate that modular automation provides both high-quality and high-yield T-cell products.MethodsCells from three individual donors collected on an automated continuous flow centrifugation system were successfully expanded in a functionally closed, automated, perfusion-based hollow fiber bioreactor. These cells were then prepared for cryopreservation in an automated closed-system device that maintains temperature and aliquots a mixed cell product and cryoprotectant into product bags. Cell product bags were thawed and expanded in flasks. Samples taken throughout this manufacturing process were analyzed for cell phenotype, exhaustion markers and functionality. The proportion of CD4+ and CD8+ T cells was maintained through each step, from pre-expansion and post-expansion to immediately after thaw and 24 h after thaw.ResultsInterestingly, phenotypic markers such as CD45RO, CD45RA and CCR7 evolved throughout the process and stem-like memory T cells emerged as the predominant phenotype in the clinically relevant 24-h post-thaw sample.ConclusionsModular automation supported the generation of stem-like memory T cells that were not terminally exhausted and were able to produce effector cytokines upon restimulation.     

Ultrasound Enhances Retrovirus‐Mediated Gene Transfer

Abstract

Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus‐mediated gene transfer efficiency.

Retrovirus‐mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with β‐galactosidase (β‐Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm² to 4.0 watts/cm²) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated.

Below 1.0 watts/cm² and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm² of an ultrasound resulted in significant increases in retrovirus‐mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6‐fold, 4.8‐fold, 2.3‐fold, and 3.2‐fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, β‐Gal activities were also increased by the retrovirus with ultrasound exposure in these cells.

Adjunctive ultrasound exposure was associated with enhanced retrovirus‐mediated transgene expression in vitro. Ultrasound associated local gene therapy has potential for not only plasmid‐DNA‐, but also retrovirus‐mediated gene transfer.