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Abstract

Chimeric antigen receptor (CAR) T-cell therapies have provided life-saving alternatives when conventional blood cancer treatments were not successful. This therapy is based on genetically modifying T cells to express CAR, to increase their ability to recognize and kill cancer cells. Despite great success and FDA approval of six commercial CAR T-cell therapies, the availability to patients remains limited in part due to complex and costly biomanufacturing. To ensure economical manufacturing of safe and effective cell therapies, the overall aim of this research was to improve process understanding and to develop appropriately tailored manufacturing technologies. To avoid unpredictable impacts on product quality during concentrated cell washing, the effects of transiently extreme microenvironment conditions in concentrated T cells were investigated. It was found that maintaining concentrated T cells at 37°C for as little as 3 h resulted in significant cell losses and impacted subsequent T-cell expansion and quality. These negative impacts were greatly diminished at room temperature for up to 3 h and at 4°C for 6-h concentrated incubation. Given these discovered tolerances to concentrated conditions, their applications in other process steps were explored. Genetic engineering of T cells to express CAR is commonly achieved using viral vectors, that increase both the costs and safety concerns. Non-viral alternatives may be preferred but a major barrier is their far lower gene delivery efficiency. This research demonstrated that T-cell transfection efficiency (i.e., transfected cells per ng mRNA) using mRNA encapsulated in lipid nanoparticles was greatly increased in concentrated cells. The improved non-viral transfection provides an alternative for a wide range of cell therapy applications. Lastly, an innovative cell processing technology that uses acoustic forces to concentrate cells was applied to cell washing, the most frequent process step in CAR T-cell therapy manufacturing. This was performed at high efficiency and an optical-based sensor was developed to monitor the device performance in real time, thus enabling automated device control as a means to address the variability of cell therapy manufacturing. Overall, the results of this research should enable improved treatment safety and reduced manufacturing costs, making life-saving cell therapies more widely available to patients in need.