39 research outputs found
Modification of T lymphocytes with lentiviral vectors for expression of anti-CD19 chimeric antigen receptor (CAR)
The use of immunotherapy with modified T lymphocytes with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemias and lymphomas resistant to chemotherapy. CAR possess an extracellular domain derived from variable regions of antibodies and costimulation intracellular domains of T lymphocytes. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors as well as normal B cells, but not in other types of cells. Recent clinical studies involving anti-CD19 CAR T-cells have shown excellent responses in a variety of B-cell tumors, even in patients with relapse after high-dose chemotherapy. This study aimed to produce CD4+ lymphocyte lineage Jurkat (ATCC® TIB-152 ™) modified with a second generation anti-CD19 CAR with 4-1BB as intracellular costimulation domain. Lentiviral vectors were produced in HEK293T (ATCC® CRL-3216 ™) transiently transfected with plasmids containing the coding sequence of the CAR, viral envelope VSV-G, and viral capsid. The viral titer was calculated by real time PCR after transduction of HEK293T cells, resulting in 1.65 x 105 IU/mL. The literature indicates an MOI (multiplicity of infection) from 5 to 10 IU/cell for transduction of lymphocytes. A new batch of virus was produced, and the supernatant was ultracentrifuged at 19200 rpm (Beckman Coulter, SW28 rotor) in order to concentrate the viral particles. The viral titer of the concentrated batch was 1.26 x 108 IU/mL. This new titer is compatible with the necessary to infect 107 cells, amount of pre-expansion cells necessary to obtain the number of cells suitable for infusion into patients (2.5 x 109 to 5 x 109 cells). Then, the infection of Jurkat was performed in a 6-well plate with RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 µg/mL Polybrene®, and centrifugation at 1000 rpm for 20 minutes at room temperature. After 16 hours of incubation (37°C, 5% CO2 and 85% humidity), the medium was exchanged for fresh RPMI 1640 10% FBS. After additional 48 hours of incubation under the same conditions, the cells were collected and was their DNA was extracted. We obtained by real-time PCR that the number of integrated viral copies per genome was 35.3 ± 4.5 (mean ± standard deviation) for transduction with MOI of 5 IU/cell. While for MOI of 10 IU/cell, it was obtained 42.6 ± 0.1 copies per genome. It was observed that there was not a significant increase in viral copies when the MOI increased from 5 to 10. This may occur because cell’s surface receptors have been saturated by the large number of viruses. The lentiviral vector used by us has been shown to transduce T lymphocyte satisfactorily. The next steps of the study are the transduction of T lymphocytes from healthy donors and verification of the CAR receptor effectiveness to bind to CD19 of cell B lymphocyte lineages. Grant #2016/08374-5, São Paulo Research Foundation (FAPESP)
Experimental and economic evaluation of different culture systems for mesenchymal stromal/stem cell expansion for clinical applications
The translation of cell therapies into clinical practice requires a scalable, efficient and cost-effective manufacturing process. This study presents an integrated experimental and cost analysis of different cell culture technologies for commercial manufacture of a novel umbilical cord-derived cell therapy, currently in early phase clinical trials for the treatment of acute graft-versus-host disease (aGvHD). The experimental analysis assessed the expansion and harvest potential of mesenchymal stromal cells (MSCs), derived from umbilical cord matrix (UCM-MSCs), in different scalable cell culture technologies: a multi-layer vessel (ML), a stirred tank bioreactor with microcarrriers (STR), a hollow fiber bioreactor (HF) and a packed-bed bioreactor (PB). The presentation will highlight differences in cell proliferation rate, expansion fold and harvesting efficiency across the technologies. The cells retained their functional properties post culture in all the technologies evaluated. The experimental results were incorporated into a bioprocess economics tool comprising a stochastic cost of goods (COG) and sizing model to evaluate the commercial economic feasibility and robustness of the technologies. The financial and risk rank orders predicted by the tool will be presented, as well as their sensitivity to the reimbursement scenario selected. The model determined industrially relevant scenarios for which no technology will yield a satisfactory gross margin, indicating that many studies are still needed to establish an optimized manufacturing process
Expansion strategies for human mesenchymal stromal cells cultured under xeno-free conditions
Choosing the culture system and culture medium used to produce cells are key steps towards a safe, scalable and cost-effective expansion bioprocess for cell therapy purposes. Traditionally, mesenchymal stromal cells (MSC) have been cultured in a defined basal medium (DMEM or alpha-MEM) supplemented with fetal bovine serum (FBS). Although effective, the use of FBS or other animal-derived components in manufacturing processes is highly discouraged by regulatory agencies due to the risk of transmitting xenogeneic infectious agents and immunization. The use of AB human serum (AB HS) as an alternative xeno-free medium supplement for MSC has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix-derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno-free bioprocess. UCM MSC were cultured in a scalable planar (compact 10-layer flasks and roller bottles) and 3D microcarrier-based culture systems (spinner flasks and stirred tank bioreactor). 10 layer-flasks and roller bottles enabled the production of 2.6±0.6x104 and 1.4±0.3x104 cells/cm2 (4.5±1.1x107 and 3.0±0.7x107 cells) representing a fold increase of 13.0 and 7.11, respectively. UCM MSC-based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.0-23.0x104 cells/cm2) when compared to planar systems. Nevertheless, due to the moderate harvest efficiency attained (80% for spinner flasks and 53.9% for bioreactor) the total cell number recovered, 3.3-3.7x107 cells, was lower than expected. After the expansion in the culture systems evaluated, the cells maintained the functional properties: the cells were able to differentiate into adipocytes, chondrocytes and osteocytes and presented a typical immunophenotype profile. The cryopreservation of cells (also using AB HS) was also successfully carried out in plastic bags to facilitate post-thaw manipulation and enable clinical use. No significant difference was observed when comparing pre-cryopreservation and post-thaw viabilities of cells expanded in the planar (T-flasks) and 3D culture system (spinner flask with microcarriers) indicating that cells which expanded on a larger scale in microcarrier-based suspension cultures could be successfully cryopreserved in plastic bags, with a higher cell number (up to 4x108 cells) and under xeno-free conditions. Overall, our results indicated that among the planar culture systems evaluated, compact multilayer vessels could be used to easily produce enough cells to treat a small number of patients, and are therefore recommended for autologous therapies. However, for a fully-controlled and larger-scale cell production, in an autologous and/or allogeneic scenario, stirred tank bioreactors combined with microcarrier technology and xeno-free media can be successfully used, allowing for GMP-compliant expansion
Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII
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Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C.\ud
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Results\ud
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We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection.\ud
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Conclusion\ud
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Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.The authors would like to acknowledge FAPESP (2008/51505-7) and FINEP (01.07.0652.00) for financial support
Evaluating kinetic and physiological features of rCHO-K1 cells cultured on microcarriers for production of a recombinant metalloprotease/disintegrin
Evaluating kinetic and physiological features of rCHO-K1 cells cultured on microcarriers for production of a recombinant metalloprotease/disintegrin
We present kinetic and physiological data regarding the culturing of
rCHO-K1 cells on various microcarriers, to evaluate the potential of
this culture strategy for mass production of these cells and expression
of a recombinant disintegrin. Cultures were performed in 500 mL spinner
flasks in DMEM culture medium with 10% v/v fetal calf serum, gently
shaken at 37\ub0C, pH 7.4, in a 10% v/v CO2 atmosphere. The following
values were obtained, respectively, for the adhesion time-constant Ka
(h) and specific growth rate \u3bcmax (d-1) on each microcarrier:
Cytodex 1 (0.91, 0.45), Cultispher S (0.28, 0.34), Immobasil FS (0.85,
0.52) and Pronectin F (5.12, 0.67). Metabolic characteristics showed
some variation among the cultures with the four microcarriers, the most
significant being the higher production of ammonia with microcarriers
coated with adhesive molecules (Cultispher S and Pronectin F) relative
to the uncoated carriers (Cytodex 1 and Immobasil FS). Experiments
where the DMEM medium was gradually replaced by the serum-free medium
(CHO-SFM-II) revealed important advantages over media containing serum,
not only for assay purposes, but also for purification of the
disintegrin. Altogether these results demonstrate that cultures on
microcarriers, especially on Pronectin F, show good potential for
larger scale cultures of rCHO-K1 cell
Production of Lentiviral Vectors Encoding Recombinant Factor VIII Expression in Serum-Free Suspension Cultures
Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo
IntroductionNatural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary.MethodsIn this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence.ResultsWe demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model.ConclusionTogether with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals
Mesenchymal Stromal Cells: From Discovery to Manufacturing and Commercialization
Over the last decades, mesenchymal stromal cells (MSC) have been the focus of intense research by academia and industry due to their unique features. MSC can be easily isolated and expanded through in vitro culture by taking full advantage of their self-renewing capacity. In addition, MSC exert immunomodulatory effects and can be differentiated into various lineages, which makes them highly attractive for clinical applications in cell-based therapies. In this review, we attempt to provide a brief historical overview of MSC discovery, characterization, and the first clinical studies conducted. The current MSC manufacturing platforms are reviewed with special attention regarding the use of bioreactors for the production of GMP-compliant clinically relevant cell numbers. The first commercial MSC-based products are also addressed, as well as the remaining challenges to the widespread use of MSC-derived products
Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening
Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better.Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation.Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300–500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43).Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application