Abstract
The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/β-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/β-free CAR19 T cells at clinical scale.
Highlights
A successful concept to treat hematological cancers is based on the genetic engineering of T cells to express chimeric antigen receptors (CARs)
We report on cellular composition, T cell phenotype, as well as TRAC knockout efficacy and demonstrate that a >99.5% TCRa/b-free CAR T cell product can be generated with high quality and conservation of its cytolytic potency
T cells were activated for 3 days before being nucleofected with the indicated CRISPR-Cas[9] ribonucleoprotein (RNP) complexes or with transcription activator-like effector nuclease (TALEN)-encoding mRNA
Summary
A successful concept to treat hematological cancers is based on the genetic engineering of T cells to express chimeric antigen receptors (CARs). As opposed to the natural T cell receptor (TCR), CARs bind tumor antigens or tumor-associated antigens in a human leukocyte antigen (HLA)-independent manner. Anti-CD19 CAR T cells have shown remarkable success in treating CD19-expressing B cell malignancies, with the first two CAR T cell products (Kymriah, Yescarta) approved by the FDA and EMA in 2017 or 2018, respectively. The generation of CAR T cells typically involves the delivery of a CAR transgene into autologous T cells using (semi-)randomly integrating viral or non-viral vector systems.[1] the generation of CAR T cells from autologous sources is highly dependent on the quality of the starting T cell apheresis material. 9% of the patients enrolled in the pivotal trial entailing Kymriah did not receive their
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