Background: Adoptive T-cell therapies with CD19 chimeric antigen receptors (CAR) are now the standard of care for refractory/recurrent hematologic B-cell malignancies. However, challenges remain in delivering a more fit cell product that can be manufactured in a more timely and cost-effective manner. To address these challenges, a rapid same-day fully closed manufacturing process was developed to generate a more fit product for subcutaneous administration and to enable an efficient transition from early-phase clinical trials to commercial-scale production. Methods: A lentivector (LV) was designed with the capability of directly targeting and activating T cells simultaneously to enable manufacturing without prior T-cell selection. Experiments were performed to understand the kinetics of direct lentivector-induced T-cell activation, internalization, reverse transcription, and vector genome integration. After establishing critical process parameters for efficient loading of T cells with LVs, a closed manufacturing process was developed from peripheral blood mononuclear cells (PBMC) starting material derived from whole blood or apheresis. PBMCs were isolated in an automated closed system and exposed to LV for a four-hour incubation period. Cells were washed in an automated closed system to remove residual LV, formulated, and vialed with release testing for identity, potency, purity, and safety. Three full-scale engineering lots were manufactured from donor-derived apheresis in addition to three small-scale analytical lots with patient-derived PBMC. Results: For targeted gene delivery into T cells within PBMC, the CD19 CAR LV was designed to contain a CD3 targeting motif in its envelope to enable T-cell binding, activation, and viral entry into CD3+ T cells. The CD3-directed LV encoding a CD19-CAR and a novel synthetic driver co-stimulatory domain was manufactured in a chemically defined 25L clinical scale suspension-based process and vialed to a target unit potency for ease of cell loading. The purified CD3-directed LV is capable of inducing T-cell receptor internalization and activation through specific bioassays. In surrogate analytical cultures, following LV exposure and automated washing, viral entry into T cells was complete. Analytical cultures demonstrate the ability to successfully generate CAR+ cells with a unique CD3+CD56+CD45RA+CCR7+ T memory-stem cell with NK-like features and a vector copy number of less than 5 vector copies/transduced cell (1.95 +/- 0.58). In preparation for clinical investigation, full-scale runs were performed in GMP facilities with qualified methods for release testing against identity, potency, purity, and safety. Small-scale analytical processes utilizing B-cell lymphoma patient-derived PBMC from whole blood were evaluated for differences relative to donor-derived material. Conclusions: To address current challenges in the manufacturing of autologous cell products, a same-day closed manufacturing process was developed to produce cells with enhanced fitness. Qualification of this novel design included 1) completion of the production of the CD3-directed LV encoding a CD19 CAR with a novel synthetic co-stimulatory driver element at the 25L scale; 2) completion of three full-scale closed engineering runs along with the qualification of QC release testing for identity, potency, purity, and safety and 3) comparability studies utilizing patient-derived PBMC.