Antibody‐Based CAR T Cells Produced by Lentiviral Transduction

Abstract

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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“…[80] Within the scope of T cell expansion, magnetic particle aAPCs conjugated with anti-CD28 (𝛼CD28) and anti-CD3 antibodies (𝛼CD3) have emerged as fundamental to both clinical and scientific research. [81][82][83] However, this method has limitations, such as inducing an effector state in T cells before transfusion, resulting in decreased viability. [84,85] To overcome these limitations, Roh's team enhanced T cell viability by incorporating a molecular modification of HER2 onto the surface of iron oxide particles (Dynabeads), creating a cell-free artificial target particle (aaTP) that induces CAR-T cell activation (Figure 3a).…”

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“…Pan T cells were isolated from healthy donor or CLL patient-derived whole blood (Pan T Cell Isolation Kit and Auto MACS Quant, Miltenyi, Bergisch Gladbach, Germany), stimulated with CD3/CD28 T-cell activation Dynabeads (Life Technologies, Carlsbad, USA) at a 1:1 bead to cell ratio, and lentivirally transduced 24 hours later at a multiplicity of infection of 1.5 or retrovirally transduced 48 hours after isolation 43 . All T cells were expanded in complete T cell medium supplemented with penicillin-streptomycin (100 U/mL; Life Technologies, Carlsbad, USA)) and fed IL-2 (50 U/mL; Stem Cell Technologies, Vancouver, Canada) every 48 hours.…”