High-Content CD14+ Cells in Apheresis Products negatively affects the Transduction and Expansion of CAR T cells during Large-scale cGMP Manufacturing

Abstract

Abstract Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is a promising strategy to treat patients with cancer. Apheresis products are commonly used as 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 encountered manufacturing challenges with apheresis products containing high proportion of CD14+ cells. As an illustration, we investigated the transduction and expansion of T cells using an apheresis product derived from a patient with ALL and containing 70% CD14+ cells. 350 × 106 CD3+ cells were selected from a cryopreserved apheresis product using CD3/28 magnetic beads. A short adherence procedure decreased the CD14+ cell composition from 70% to 45%. On day 3, 350×106 vs 20 ×106 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. 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%). 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 will be discussed and additional samples will be analyzed.