Optimized High-Titer Lentivirus Production and Efficient CAR-T Cell Generation

Chimeric antigen receptor (CAR)-T cell treatment has shifted the paradigm of cancer immunotherapy, especially in B-cell malignancies, with around an 80% response rate in acute lymphoblastic leukemia. Emerging data on the use of CAR-T cells in the treatment of autoimmune diseases, such as lupus, indicate another significant advance in this therapeutic field and highlight its promising potential for the future. Despite its remarkable therapeutic potential, the manufacturing of CAR-T cells, including the production of critical components such as high-titer lentiviral vector (LV), remains a significant challenge, limiting the broader accessibility and the ease of manufacturing and administration of this therapy. Lack of optimized and harmonized protocols makes the manufacturing of CAR-T cells cumbersome. To ease some of the challenges of CAR-T cell manufacturing, we optimized LV production, with an emphasis on enhancing viral titers using different transfer-to-helper plasmid ratios, optimizing transfection efficiency in HEK293T cells, and fine-tuning peripheral blood mononuclear cell activation and transduction conditions. We present a set of optimized protocols for the scalable production of third-generation LVs for CAR-T cell generation. This approach facilitates preclinical and translational research by providing a framework for the reproducible, economical, and effective production of CAR-T cells. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Plasmid ratio screening Basic Protocol 2: Viral concentration via ultracentrifugation Basic Protocol 3: FACS-based titration assay Basic Protocol 4: Peripheral blood mononuclear cell (PBMC) isolation Basic Protocol 5: PBMC activation for effective transduction Basic Protocol 6: CAR-T cell generation with lentivirus Support Protocol: Flow cytometry gating strategies.