Enrichment of CD4+ and CD8+ T lymphocytes with a column-free flow-based device for clinical cell manufacturing

Abstract

In recent years, adoptive T cell-based immunotherapies have been developed to treat a wide range of hematologic malignancies, including relapsed or refractory non-Hodgkin lymphoma, B-cell leukemia, and multiple myeloma. Most of the commercially approved adoptive T cell therapies are composed of chimeric antigen receptor (CAR)–based T cells, which are a patient's own T cells engineered for recognition of a specific surface antigen, such as CD19 or CD20. Unselected peripheral blood mononuclear cells (PBMCs) have recently been used in several manufacturing protocols, but the vast majority of protocols still use CD4/CD8-selected T cells. The first step in manufacture of these CAR-T products involves simultaneous selection/purification of CD4⁺ and CD8⁺ (or CD4/CD8 positive) T cells. The typical approach for selection of CD4/CD8 subsets for clinical manufacturing involves immunomagnetic labeling followed by selection of positively labeled cells using static column-based approaches that are prone to cell clogging events and typically take approximately 2 to 3 hours in a closed system. Here, we used a new column-free, flow-based, fully closed system suitable for clinical cell manufacturing for isolation of CD4/CD8 cells with high purity in a rapid fashion that could accommodate varying capacities without compromising cell recovery. This new approach allows markedly faster cell selection, preserving the quality of the cells that are used for downstream CAR-T cell manufacture. We report the results of our successful validation runs using the new MARS Bar enrichment platform using human apheresis-derived leukocytes for CD4/CD8 isolation in a selection buffer or directly in T cell culture media for subsequent CAR-T cell production. Our data show a rapid and robust CD4/CD8 enrichment with an enrichment time shortened to 1 hour or less. Overall purity (based on CD3⁺ expression) of the cells was 95.51 ± 1.23% and 93.13 ± 0.30% for fresh and thawed T cells, respectively. Cell recoveries were 64.68 ± 14.05% and 57.06 ± 6.28% for fresh and thawed cells, respectively. We then further tested the MARS Bar enrichment platform after cell wash/volume reduction using the LOVO Automated Cell Processing System, leading to a higher consistency in CD3⁺ purity and increased cell recovery of 68.50 ± 3.54%. Enriched cells were characterized by high viability, ie. 90.5 ± 0.05% for fresh leukopaks when used together with the LOVO device. Altogether, the new approach using the MARS Bar platform allows one to customize and standardize the selection process by using a stand-alone instrument in a clinical manufacturing setting together with cGMP grade reagents and buffers.