Optimized Production of Lentiviral Vectors for CAR-T Cell

Moço, Pablo Diego; Abreu-Neto, Mário Soares; Fantacini, Daianne Maciely Carvalho; Picanço‐Castro, Virgínia

doi:10.1007/978-1-0716-0146-4_5

Cited by 8 publications

(8 citation statements)

References 17 publications

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“…A mandatory prerequisite for conducting this protocol is the availability of lentiviral particles encoding for the CAR transgene. The underlying plasmid, which can be either self‐assembled or purchased (e.g., Creative Biolabs) has to be packaged in viral particles (Lana & Strauss, 2020; Moco, de Abreu Neto, Fantacini, & Picanco‐Castro, 2020) or the virus particles for certain CAR constructs can be purchased directly (e.g., G&P Biosciences, Creative Biolabs).…”

Section: Strategic Planning (Optional)mentioning

confidence: 99%

Antibody‐Based CAR T Cells Produced by Lentiviral Transduction

2020

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One promising approach to treat hematologic malignancies is the usage of patient‐derived CAR T cells. There are continuous efforts to improve the function of these cells, to optimize their receptor, and to use them for the treatment of additional types of cancer and especially solid tumors. In this protocol, an easy and reliable approach for CAR T cell generation is described. T cells are first isolated from peripheral blood (here: leukoreduction system chambers) and afterwards activated for one day with anti‐CD3/CD28 Dynabeads. The gene transfer is performed by lentiviral transduction and gene transfer rate can be verified by flowcytometric analysis. Six days after transduction, the stimulatory Dynabeads are removed. T cells are cultured in interleukin‐2 conditioned medium for several days for expansion. There is an option to expand CAR T cells further by co‐incubation with irradiated, antigen‐expressing feeder cell lines. The CAR T cells are ready to use after 10 (without feeder cell expansion) to 24 days (with feeder cell expansion). © 2020 The Authors. Basic Protocol: Generation of CAR T cells by lentiviral transduction

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Section: Strategic Planning (Optional)mentioning

confidence: 99%

Antibody‐Based CAR T Cells Produced by Lentiviral Transduction

2020

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“…In the case of lentiviral vectors used for therapeutic applications such as CAR-T cell therapy, the expressed CAR construct is detected by a fluorophoreconjugated antibody during flow cytometry analysis or by measuring the mRNA expression of the transgene with RT-qPCR [19,21]. These assay formats are multi-step processes, generally time consuming and results are obtained after four to five days [21,22]. Furthermore, both methods lack the ability for high-throughput sample processing that can be easily implemented at a relatively low cost.…”

Section: Introductionmentioning

confidence: 99%

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

Labisch

Wiese

Barnes³

et al. 2021

PLoS ONE

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The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity.

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“…Virus-mediated gene transfer is widely used in the preparation of CAR-T cells because viral vectors can effectively transfer genes to a variety of cell types and can stably integrate into their genomes, leading to long-term gene expression, which persists in progeny cells (8). However, the infection conditions for preparing CAR-T cells from viruses vary greatly (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

Section: Introductionmentioning

confidence: 99%

Infection Temperature Affects the Phenotype and Function of Chimeric Antigen Receptor T Cells Produced via Lentiviral Technology

Jin

Zhang

et al. 2021

Front. Immunol.

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Chimeric antigen receptor (CAR)-T cell therapy has become an important method for the treatment of hematological tumors. Lentiviruses are commonly used gene transfer vectors for preparing CAR-T cells, and the conditions for preparing CAR-T cells vary greatly. This study reported for the first time the influence of differences in infection temperature on the phenotype and function of produced CAR-T cells. Our results show that infection at 4 degrees produces the highest CAR-positive rate of T cells, infection at 37 degrees produces the fastest proliferation in CAR-T cells, and infection at 32 degrees produces CAR-T cells with the greatest proportion of naive cells and the lowest expression of immune checkpoints. Therefore, infection at 32 degrees is recommended to prepare CAR-T cells. CAR-T cells derived from infection at 32 degrees seem to have a balance between function and phenotype. Importantly, they have increased oncolytic ability. This research will help optimize the generation of CAR-T cells and improve the quality of CAR-T cell products.

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Optimized Production of Lentiviral Vectors for CAR-T Cell

Cited by 8 publications

References 17 publications

Antibody‐Based CAR T Cells Produced by Lentiviral Transduction

Antibody‐Based CAR T Cells Produced by Lentiviral Transduction

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

Infection Temperature Affects the Phenotype and Function of Chimeric Antigen Receptor T Cells Produced via Lentiviral Technology

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