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1 and ?and2).2). process steps from an open system to E7080 (Lenvatinib) functionally closed system operations in order to minimize the risk of microbial contamination, and standardizing additional process steps in order to maximize process consistency. This study reports a procedure for generating CD19 CAR-T cells in 6 days, using a functionally closed manufacturing process and defined, serum-free medium. This method is able to produce CD19 CAR-T cells that are phenotypically and functionally indistinguishable from cells produced for clinical trials by the previously described production process. genetic modification and expansion to therapeutically useful numbers. The method previously used to generate CD19 CAR-T cells at the Surgery Branch of the NCI consisted of a 10-day process that utilized open-tissue culture vessels and required human serum (HS).11,19C21 As anti- CD19 CAR-T cell therapy moves through clinical development, a more controlled system to modify genetically, expand, and harvest T cells in the absence of serum will be required. This process will need to be a robust, simple, and GMP-compliant SHFM6 production platform to scale out manufacturing, allowing multiple patient treatments to be produced simultaneously in a single facility. This article describes such a functionally closed system for producing CD19 CAR-T cells reproducibly in 6 days. Methods Clinical retroviral vector production A plasmid encoding the CD19 CAR consisting of the mouse stem-cell virus gamma-retroviral backbone engineered to express an ScFv derived from the mouse anti-CD19 hybridoma, FMC63, fused to intracellular domains from human CD28 and CD3 zeta was used for retroviral vector production. Clinical grade retroviral vector was manufactured in accordance with current good manufacturing practices (cGMP) for Phase I by the Surgery Branch Vector Production Facility, NCI, National Institutes of Health.22,23 All studies were approved by the Institutional Review Board of the National Institutes of Health. Cell culture medium As part of a medium optimization strategy, four different media and one serum-free supplement were evaluated during the process optimization: OpTmizer? CTS, AIM V (Life Technologies, Grand Island, NY), X-VIVO 15 (Lonza, Walkersville, MD), and TexMACS GMP (Miltenyi Biotec, Cambridge, MA). Individual media were evaluated with or without 2.5C5% T-cell serum replacement (TCSR), later renamed CTS? ImmuneCellSR (Life Technologies). Where indicated, AIM V medium containing either 1% or 5% HS was used as positive controls. All media contained the following: Glutamax-1 (100??; Life Technologies), Pen/strep (100??; Lonza), and hIL-2 (300?IU/mL; Prometheus, San Diego, CA). For activation of T cells with anti-CD3 antibody, OKT3 (Miltenyi Biotech) was added to each medium at a final concentration of 50?ng/mL. Ficoll isolation of PBMCs and cell wash steps The Sepax II E7080 (Lenvatinib) cell processing device from Biosafe America (Houston, TX) is an automated closed system for processing of blood-derived cell products. E7080 (Lenvatinib) Apheresis products from healthy donors and clinical trial subjects were concentrated and their volume reduced to 120?mL using the Sepax PeriCell program and a CS-490.1 kit. PBMCs were isolated from volume-reduced apheresis products using the Sepax NeatCell program with Ficoll (Lonza) density gradient centrifugation and a CS-900.2 kit in accordance with the manufacturer’s instructions. Sepax CultureWash program and the CS-600.1 kit was used for cell culture washes after the activation step and for the final cell product wash. Manual processing of apheresis products was carried out as described previously.24 Transduction, expansion, and cryopreservation of CD19 E7080 (Lenvatinib) CAR-T cells PBMCs were collected from the apheresis products of healthy donors and melanoma or lymphoma patients using the Sepax II for automated closed system processing of blood-derived cell products (Biosafe America). Freshly processed PBMCs (day 0) were cultured in PermaLife bags (OriGen Biomedical, Austin, TX) at 1??106 cells/mL (1??109 cells in 800?mL of Optimizer medium) and activated with anti-CD3 antibody (OKT3; 50?ng/mL) and human IL-2 (300?IU/mL) for 2 days. On day 1, new PermaLife bags were coated with retronectin (GMP-grade recombinant human fibronectin fragment CH296; Takara BioDivision, Shiga, Japan) at 10?g/mL diluted in phosphate-buffered saline (PBS) and stored at 4C overnight. The next day, the retronectin was removed and the bags washed once with 2.5% HEPES in HBSS (Lonza). In some experiments, the retronectin was removed and bags were blocked with 2.5% human serum albumin (HSA) in PBS for 30?min prior to the wash. Retroviral vector supernatant.