P09.08 Clinical-grade manufacturing of ROR1 CAR T cells using a novel virus-free protocol. (1st October 2020)

• Record Type: Journal Article
• Authors: Mestermann, K; Eichler, M; Machwirth, M; Kebbel, K; Köhl, U; Einsele, H; Müller, C; Lehmann, J; Raskó, T; Lundberg, F; Izsvák, Z; Schmiedeknecht, G; Hudecek, M
• Is Part Of: Journal for immunotherapy of cancer. Volume 8(2020)Supplement 2
• Page Start: A55
• Page End: A56
• Publication Date: 2020-10-01
• Subjects: Cancer -- Immunotherapy -- Periodicals; Cancer -- Immunological aspects -- Periodicals; Tumors -- Immunological aspects -- Periodicals; Immunotherapy -- Periodicals; 616.99406105
• DOI: 10.1136/jitc-2020-ITOC7.108
• Languages: English
• ISSNs: 2051-1426

Abstract

Abstract : 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.