Background Immunotherapy with T cells that were modified by gene-transfer to express a ROR1-specific chimeric antigen receptor (ROR1 CAR-T) has therapeutic potential in ROR1⁺ malignancies in hematology and oncology. The ROR1 tumor antigen has a favorable expression profile with absence in vital normal human tissues. In this study, we sought to establish and validate clinical-grade manufacturing of ROR1 CAR-T to enable a Phase I/IIa clinical trial. In particular, we sought to integrate virus-free gene-transfer based on Sleeping Beauty transposition into this manufacturing protocol to permit scale-up and export to point-of-care manufacturing, and to reduce turn-around time, complexity and regulatory burden associated with conventional viral gene-transfer (biosafety level 2 to biosafety level 1).

Materials and Methods Buffy coats or leukaphereses were obtained from healthy donors to perform protocol optimization (n=7) and scale-up runs (n=1). CD4⁺ and CD8⁺ T cells were isolated separately by magnetic selection and stimulated with CD3/CD28 TransACT® reagent. T cells were transfected with mRNA encoding hyperactive Sleeping Beauty transposase (SB100X) and minicircle DNA (MC) encoding a pT2 transposon comprising the ROR1 CAR and an EGFRt marker gene using the MaxCyte GTx ® electroporation platform. Following transfection, T cells were expanded for 10–13 days in G-REX® bioreactors and then harvested and formulated into the drug product at a 1:1 ratio of CAR-expressing CD4:CD8 T cells. The drug product underwent comprehensive phenotypic, functional and genomic analyses as part of product qualification.

Results The set of protocol optimization runs resulted in a highly robust process. On average, the stable gene-transfer rate at the end of the manufacturing process was 71% in CD4+ (n=5) and 54% in CD8⁺ T cells (n=7). The average yield of ROR1 CAR-T relative to the number of input T cells was 12.6-fold for CD4⁺ and 9.4-fold for CD8⁺ after 12–15 days of expansion, with an average viability of 84% for CD4⁺ and 82% of CD8⁺ T cells. The scale-up run was performed with a leukapheresis product from which 52.5 × 10^6 CD4⁺ and 109 × 10^6 CD8⁺ T cells were transfected. At the end of the manufacturing process (day 12), there were 844 × 10^6 CAR-expressing CD4⁺ (~16-fold expansion) and 857 × 10^6 CAR-expressing CD8⁺ T cells (8-fold expansion). In functional testing, ROR1 CAR-T showed specific recognition and potent elimination of ROR1⁺ target cells, as well as antigen-dependent cytokine production and productive proliferation in in vitro analyses. Experiments to determine the anti-tumor potency of the drug product in vivo and detailed genomic analyses are ongoing. Preliminary analyses suggest a favorable genomic insertion profile of the CAR transposon, and a transposon copy number that is well within the range acceptable for clinical use of the drug product.

Conclusions With this novel protocol, we aim to obtain the first manufacturing license for CAR-T in Europe that integrates our optimized approach with SB100X mRNA and transposon MC for CAR gene-transfer on the MaxCyte transfection platform. The quality and yield of the drug product support the design and dose escalation of the proposed clinical trial with ROR1 CAR-T, and will serve as a blueprint for other CAR-T products from our pipeline.

Disclosure Information K. Mestermann: None. M. Eichler: None. M. Machwirth: None. K. Kebbel: None. U. Köhl: None. H. Einsele: None. C. Müller: None. J. Lehmann: None. T. Raskó: None. F. Lundberg: None. Z. Izsvák: None. G. Schmiedeknecht: None. M. Hudecek: None.