Advancing CAR-T Manufacturing Through Comprehensive Cellular Analysis and the Development of a Lentiviral Vector Platform

Abstract

The development of ATMPs, particularly CAR-T cells, represents a significant advance in the treatment of certain forms of blood and lymphoid cancers. However, the sharp increase in demand in the coming years is accompanied by bottlenecks in manufacturing capacity and infrastructure, as well as a growing financial burden on healthcare systems. Against this backdrop, this work addresses two key questions regarding decentralized clinical feasibility. Part one established core elements for a clinical lentiviral vector production platform including reproducible serum-free suspension seed train and transfection including bioreactor production. Targeted overgrowth on the day of transfection was shown to alter cell cycle and pH, and consequently the stability and titer during subsequent processing. Combinatorial purification steps were tested, resulting in the establishment of a filtration process excluding centrifugation employing scalable tangential flow filtration. Part two involved an experimental design: three Prodigy Good Manufacturing practice (GMP) CAR-T cell manufacturing runs in a cleanroom using healthy donor material, accompanied by daily sampling. Intracellular changes were detected using a panel of more than 130 analytes using DigiWest™ technology, accompanied by flow cytometry panel to investigate extracellular markers phenotypes. By establishing a combined cytotoxicity and tumor re-challenge in vitro model, effector function and responsiveness to repeated stimulation could be monitored in a time-resolved manner. For the first time, the temporal evolution of key biological processes, including T-cell activation, CAR signaling dynamics before and after tumor contact and metabolic reprogramming, was characterized throughout a complete GMP-compliant CAR-T manufacturing process. The results revealed clearly distinguishable functional phases, with a critical transition occurring between days 5 and 7. Thus, this work contributes to the further development of decentralized manufacturing strategies for ATMPs and establishes a practical insight for patient-oriented GMP-related application in academic and clinical production environments.