Original Assignee Australian Stem Cell Centre Ltd Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.) ( en Inventor Andrew George Elefanty Edouard Guy Stanley Elizabeth Siewsun Ng Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.) Granted Application number US12/756,785 Other versions US10894944B2 ![]() Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies.- Google Patents US20100317104A1 - Cell culture mediaĭownload PDF Info Publication number US20100317104A1 US20100317104A1 US12/756,785 US75678510A US2010317104A1 US 20100317104 A1 US20100317104 A1 US 20100317104A1 US 75678510 A US75678510 A US 75678510A US 2010317104 A1 US2010317104 A1 US 2010317104A1 Authority US United States Prior art keywords serum media composition free media differentiation free Prior art date Legal status (The legal status is an assumption and is not a legal conclusion. ![]() CAR T cell final products met release criteria, except for one showing myc overexpression and another with viral copy number higher than five. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency of Medicines and Medical Devices (AEMPS) for the manufacture of investigational advanced therapy medicinal products (ATMPs). NKG2D CAR T cells expanded between 10 and 13 days. Then, we used NKG2D-CD8TM-4-1BB-CD3ζ lentiviral vector for cell transduction (MOI = 2). A total of 108 CD45RA- cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. CD45RA+ fraction was depleted from healthy donors' non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. ![]() To avoid such adverse effects, we used CD45RA- memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients' own T cells, disease progression, and death prior to return of engineered cells. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3ζ signaling domain. NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. ![]() Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors.
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