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

<h3>Background</h3> 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<sup>+</sup> 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 <i>Sleeping Beauty</i> 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). <h3>Materials and Methods</h3> Buffy coats or leukaphereses were obtained from healthy donors to perform protocol optimization (n=7) and scale-up runs (n=1). CD4<sup>+</sup> and CD8<sup>+</sup> 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. <h3>Results</h3> 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<sup>+</sup> 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<sup>+</sup> and 9.4-fold for CD8<sup>+</sup> after 12–15 days of expansion, with an average viability of 84% for CD4<sup>+</sup> and 82% of CD8<sup>+</sup> T cells. The scale-up run was performed with a leukapheresis product from which 52.5 × 10^6 CD4<sup>+</sup> and 109 × 10^6 CD8<sup>+</sup> T cells were transfected. At the end of the manufacturing process (day 12), there were 844 × 10^6 CAR-expressing CD4<sup>+</sup> (~16-fold expansion) and 857 × 10^6 CAR-expressing CD8<sup>+</sup> T cells (8-fold expansion). In functional testing, ROR1 CAR-T showed specific recognition and potent elimination of ROR1<sup>+</sup> target cells, as well as antigen-dependent cytokine production and productive proliferation in <i>in vitro</i> analyses. Experiments to determine the anti-tumor potency of the drug product <i>in vivo</i> 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. <h3>Conclusions</h3> 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. <h3>Disclosure Information</h3> <b>K. Mestermann:</b> None. <b>M. Eichler:</b> None. <b>M. Machwirth:</b> None. <b>K. Kebbel:</b> None. <b>U. Köhl:</b> None. <b>H. Einsele:</b> None. <b>C. Müller:</b> None. <b>J. Lehmann:</b> None. <b>T. Raskó:</b> None. <b>F. Lundberg:</b> None. <b>Z. Izsvák:</b> None. <b>G. Schmiedeknecht:</b> None. <b>M. Hudecek:</b> None.