Demonstrating the Manufacture of Human CAR-T Cells in an Automated Stirred-tank Bioreactor.

Chimeric antigen receptor T-cell (CAR-T) therapies have proven clinical efficacy for the treatment of haematological malignancies, with successful long-term patient outcomes. In spite of the clinical promise however, many of the commercially-approved or late-stage clinical trial therapies are prohibitively expensive to manufacture. Consistent and scalable manufacturing technologies are required to isolate, engineer and expand the cells to reduce the cost of these patient-specific therapies. In this study, we demonstrate, for the first time, the manufacture of human CAR-T cells from multiple donors in an automated stirred-tank bioreactor. Building on previous work with primary T-cells, we not only demonstrate the successful production of functional human CAR-T cells from multiple donors under dynamic conditions in a stirred-tank bioreactor, but that overall cell yields were significantly better than in static T-flask culture cultured over the same time period. We found that at agitation speeds of 200 rpm (specific power of 74 × 10-4 W kg-1 ) and greater (up to 500 rpm, specific power of 1164 × 10-4 W kg-1 ), the CAR-T cells were able to proliferate effectively, reaching viable cell densities of > 5 × 106 cells ml-1 over a period of 7 days. Such values are comparable with current expansion systems and significantly better than static expansion platforms such as T-flasks and gas-permeable culture bags. Importantly, the engineered T cells post-expansion in the stirred-tank bioreactor retained expression of the CAR gene. The CAR-T cells also retained their cytolytic function even when grown at the highest agitation intensity (500 rpm, P/M = 1164 × 10-4 W kg-1 ), thereby proving that power inputs used in this study do not affect CAR-T cell efficacy to target and kill the leukaemia cells in vitro. This assessment of product quality was reinforced by the cytokine release assay where it was demonstrated that substantial secretion of proinflammatory cytokines (IL-2, TNF, and INF-γ) was detected after 24 hours of co-culture with NALM6 cells (CD19 expressing cells), showing no significant difference between the assessed conditions. This study is the first demonstration of human CAR-T cell production in stirred-tank bioreactors with the findings presenting significant implications and opportunities for larger-scale allogeneic CAR-T production. This article is protected by copyright. All rights reserved.