Programmpunkt

12:30

Titel: Automated generation of gene-edited CAR T cells at clinical scale
ID: VS-5-4
Art: Abstractvortrag
Redezeit: 5 + 5 min
Session:
Sektion Stammzelltransplantation und Zelltherapie
VS-5

Referent: Jamal Alzubi (Freiburg)


Abstract - Text

Offenlegung Interessenkonflikt:

DL, SW, TS and AK are employees of Miltenyi Biotec. TCa and TIC have sponsored research collaborations with Miltenyi Biotech and Cellectis.

Methods

A fully automated process that unites lentiviral CAR delivery with gene editing to generate allogeneic off-the-shelf CAR T cell products would help to overcome these limitations. As a proof-of-concept, we combined lentiviral delivery of a CD19-targeting CAR transgene with designer nuclease mediated knockout of the endogenous TRAC locus, which codes for T-cell receptor (TCR) alpha chain. The extended CliniMACS Prodigy platform contains an in-line electroporation unit that was used to deliver a TRAC targeting TALEN designer nuclease in the form of mRNA.

Results

At clinical scale, the TRAC locus was disrupted in more than 35% of cells with high cell viability (>90%) and minimal off-target activity. Upon a negative selection step, the established automated process enabled us to generate a CAR T cell product with >99% TCR-free cells. These TCR-knockout CAR T cells proliferated well and maintained a T cell memory phenotype. Similar to the unedited CAR T cells, the TCR-free CAR T cells were able to eliminate CD19-positive tumor cells and to release the expected cytokines when challenged with B cell leukemia cells.

Background

Chimeric antigen receptor (CAR) T cell immunotherapy has shown remarkable success in the treatment of patients with hematologic malignancies. The manufacturing of CAR T cells in combination with targeted gene disruption, however is a rather complex and expensive process. The several open manufacturing steps and patient-to-patient variation in the quality of the apheresis material are associated with risks of contamination and manufacturing failure due to poor expansion of the patient"s T cells.

Conclusion

In conclusion, we established a fully automated, GMP-compliant manufacturing process that integrates lentiviral transduction with electroporation of TALEN mRNA in order to produce fully functional TCR-free CAR T cells at clinical scale. Such protocols will facilitate the decentralized production of off-the-shelf CAR T cell products and their successful clinical application.