A new sort of cells for chimeric antigen receptor T-cell therapies-isolating CD14^-CD127⁺ T cells for chimeric antigen receptor T-cell manufacture.

Abstract

Chimeric antigen receptor (CAR) T cell therapy is an adoptive immunotherapy in which T cells are isolated from the patient and genetically modified ex vivo to express a CAR, thus enabling the CAR T-cells to target tumor cells. Currently, six Food and Drug Administration-approved CAR T-cell therapies target either CD19 for relapsed/refractory (R/R) B cell malignancies or B cell maturation antigen for R/R multiple myeloma. The success of these CAR T-cells has positioned this therapy as an important option for treating hematological malignancies and has also spurred efforts to use CAR T-cells to target solid tumors and to target nonmalignant B cell pathologies. The final functionality of CAR T-cells is influenced by the initial T cell subpopulations that were isolated, and naïve and memory phenotypes have been shown to yield superior final products. Our goal is to refine the T cell isolation method to enrich a naïve/memory T cell population for CAR T-cell production, while excluding cell subsets that could impair CAR T-cell product performance. Healthy donor PBMCs were subjected to magnetic cell separation to deplete CD14⁺ cells, followed by a positive selection of CD127⁺ cells. The enriched CD14^-CD127⁺ T cell population was characterized prior to CAR T-cell production. For comparison, we also generated CAR T-cells using CD14^-CD25^-CD62L⁺ T cells in an established method. Both CAR T-cell products were evaluated for production quality and compared in a series of activity assays. A new isolation method was applied to healthy donor PBMCs to enrich a CD14^-CD127⁺ T cell population. With this approach, B cell populations and monocytes were largely excluded from the final cell population, and extremely low percentages of CD4+FoxP3+ regulatory T cells (Tregs) were detected. The enriched CD14^-CD127⁺ T cell population was largely comprised of naïve, stem-like central memory, and central memory T cells. A BAFF-R targeted CAR (MC10029) was transfected into both CD14^-CD25^-CD62L⁺ T cells and CD14^-CD127⁺ T cells from the same donor. Both CAR T-cell products exhibited similar cytotoxic performance. We developed a new two-step isolation method for enriching CD14^-CD127⁺ T cells from PBMCs. This method effectively excluded unwanted B cells, yet maintained the naïve, stem-like central memory and central memory T cells that can produce functional CAR T-cells.