80. Depletion of High-Content CD14+ Cells from Apheresis Products is Critical for the Successful Transduction and Expansion of CAR T Cells During Large-Scale cGMP Manufacturing

Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells holds great promise as a novel strategy to treat patients with cancer. Apheresis products are a major source of starting material for large-scale T cell selection using CD3/28 magnetic beads. At the time of collection, the composition in various cell lineages is highly variable. We have successfully manufactured CAR T cell products starting from apheresis products containing as few as 0.5% to 3% of CD3+ cells. However, we have experienced manufacturing challenges with apheresis products containing a high fraction of CD14+ cells. We conducted a study with an apheresis product derived from a patient with acute lymphoblastic leukemia containing 70% CD14+ cells. 350 × 106 CD3+ cells were selected from the cryopreserved apheresis product using CD3/28 magnetic beads either with or without a prior 1.5 hr monocyte/granulocyte adherence step. This short adherence procedure decreased the CD14+ cell composition from 70% to 45%. A second overnight depletion by adherence in T-flasks and re-stimulation with CD3/28 magnetic beads on day 1 were also performed in the depleted group to ensure the proper activation of CD3+ cells. On day 3, 350×106 vs 20 x106 total cells were recovered from each group without and with the depletion step, respectively. Selected cells from both groups were transduced using the same dilution of vector stocks at a cell concentration of either 0.4×106/mL or 0.2×106/mL in tissue culture bags. On day 7, we observed a drastic difference in transduction efficiency between the group without depletion (4.5%) and the group in which the CD14+ cells were depleted (38%). Moreover, starting with 108 × 106 selected cells on day 3, a mere total of 12.3 × 106 (14.8% CD3+) were recovered on day 11 in the group without depletion. By contrast, cells from the depleted group expanded 350 fold from day 3 to day 10 (100% CD3+). Our results suggest that high CD14+ cell content poses a manufacturing challenge when the selection of the CD3+ cells in the apheresis product is performed with CD3/CD28 magnetic beads. The CD14+ cell threshold for successful manufacturing without depletion remains to be determined. However, monitoring the CD14 cell content and depleting these cells from starting apheresis products with high CD14 content prior to CD3/CD28 magnetic bead selection are critical to ensure success of the CAR T cell manufacturing process.