287. Production of KTE-C19 (Anti-CD19 CAR T Cells) for ZUMA-1: A Phase 1/2 Multi-Center Study Evaluating Safety and Efficacy in Subjects with Refractory Aggressive Non-Hodgkin Lymphoma (NHL)
Authors: Marc Better, Vijay Chiruvolu, James Oliver, Emily Lowe, John M. Rossi, Arianne Perez, Lynn Navale, Adrian Bot
Journal: Molecular Therapy (2016)

Introduction: An ongoing anti-CD19 CAR T cell study with KTE-C19 in subjects with refractory, aggressive NHL achieved objective responses in 5/7 subjects, including 4 complete remissions (Locke, ASH 2015). To support this trial (ZUMA-1, NCT02348216) and other multicenter trials in lymphoma, we developed a robust and efficient strategy to generate autologous anti-CD19 CAR-engineered T cell products. The newly developed process aims to minimize the time between subject leukapheresis and product administration, and generate a KTE-C19 lot for every enrolled subject. Methods: Upon confirmation of eligibility, leukapheresis was performed to process 12-15 L of blood targeting collection of 5-10 × 109 peripheral blood mononuclear cells (PBMC). After collection at the investigational site, subject apheresis material was shipped to the central manufacturing site, where it was processed to enrich for the T cell-containing PBMC fraction on a closed system Ficoll™ gradient. In a closed bag system, T cells in the PBMC fraction were then activated using an anti-CD3 monoclonal antibody and cultured in serum-free medium containing 300 IU/ml of IL-2. Magnetic beads were not used for either cell selection or activation. Activated T cells were transduced with a gamma retroviral vector that contains the anti-CD19 CAR gene and further expanded for 4 to 6 days to achieve the target cell dose of 2 × 106 CAR-positive T cells/kg (minimum of 1 × 106). Final KTE-C19 product was washed, cryopreserved and tested for identity, potency, and adventitious agents. In-process samples were collected for analysis by flow cytometry for CAR expression and other characteristics. After meeting acceptance criteria, the KTE-C19 product was shipped back to the clinical sites using a validated cryo-shipper. Results: 7 subjects were dosed in the phase 1 portion of ZUMA-1. KTE-C19 was successfully manufactured in all cases despite a broad range of baseline leukocyte counts (median 5.4×103 cells/µl, 2.1-11.1) and low numbers of baseline lymphocytes (median 0.9×103 lymphocytes/µl, 0.1-1.4) prior to apheresis. Apheresed cell populations used to generate clinical lots had a broad range of phenotypic characteristics including lymphocyte / monocyte ratio (median 1.1, 0.04-3.5), CD8/CD4 ratio (median 4.2, 0.3-7.7), and naïve plus central memory (Tcm) / more differentiated T cells (median 0.3, 0.04-1.3). Fold-expansion of T cells was consistent among the 7 product lots (average 6-fold) from transduction to harvest. All KTE-C19 lots contained predominantly CD3+ T cells (median 96%; 90-99%), with CD8+ T cells (median 57%, 27-82%) and CD4+ T cells (median 43%, 18-73%). Both CD4+ and CD8+ T cells expressed CAR at similar levels. The product lots contained predominantly effector memory (median 42%, range 30-56%) and Tcm cells (median 34%, 15-58%), with the remainder being naïve (median 14%, 6-19%) and effector T cells (median 5%, 1-22%). All KTE-C19 lots met release specifications and were available for clinical administration within ~2 weeks of apheresis. Conclusions: We developed a robust KTE-C19 manufacturing process that successfully generated biologically and clinically active product irrespective of the characteristics of the starting cell populations processed to date. Product lots met release specifications and were available for subject treatment within the target timeframe. This centralized manufacturing process is well suited to support multicenter clinical trials.