Effects of starting cellular material composition on chimeric antigen receptor T‐cell expansion and characteristics

Abstract

BACKGROUND When manufacturing chimeric antigen receptor (CAR) T cells using anti‐CD3/anti‐CD28 beads, ex vivo T‐cell expansion is dependent on the composition of leukocytes used in the manufacturing process. We investigated the effects of leukocyte composition on CAR T‐cell expansion and characteristics using an alternative manufacturing method. METHODS Anti–B‐cell maturation antigen and CD19‐CAR T cells were manufactured using autologous peripheral blood mononuclear cell (PBMNC) concentrates. The PBMNCs were enriched for lymphocytes using density gradient separation, which were used for CAR T‐cell culture initiation. T‐cell expansion was stimulated with soluble anti‐CD3 and interleukin‐2. RESULTS Fifty‐one CAR T‐cell products were evaluated; 28 anti–B‐cell maturation antigen (BCMA) CAR T cells produced for 24 patients and 27 CD19 CAR T cells produced for 24 patients. CAR T‐cell expansion was reduced when greater quantities of monocytes were present in the post–density gradient separation PBMNCs. In addition, the ratio of CD4 to CD8 cells in the CAR T‐cell products after 7 days of culture was dependent on the quantity of monocytes, RBCs, and neutrophils in the post–density gradient separation PBMNCs. Greater quantities of monocytes and RBCs were associated with a greater proportion of CD4+ cells and greater quantities of neutrophils were associated with a greater proportion of CD8+ cells. CONCLUSIONS The composition of leukocytes used to manufacture CAR T cells can affect cell expansion and the composition of CAR T‐cell products. More uniform or complete lymphocyte enrichment of PBMNCs improves the consistency of final CAR T‐cell products.

Leukocytapheresis in nonmobilized donors for cellular therapy protocols: Evaluation of factors affecting collection efficiency of cells

“…30 Knowing the composition of the collected bag is also important because it affects not only the optimal storage conditions, 31 but also T-cell expansion and the characteristics of the final CAR T-cell product. 32 Our study has some limitations. It was a retrospective study and all caveats with this study design apply to our study.…”

“…Platelet contamination is a problem when monocytes are purified in vitro by adherence due to space competition; platelets bind to the monocytes, forming clumps, and reducing monocyte yield . Knowing the composition of the collected bag is also important because it affects not only the optimal storage conditions, but also T‐cell expansion and the characteristics of the final CAR T‐cell product …”

Overcoming Chimeric Antigen Receptor (CAR) Modified T-Cell Therapy Limitations in Multiple Myeloma

“…Besides, the generation of autologous CAR T-cells might be a long procedure and progression of the disease could happen during manufacturing in advance-stage cancer patients (63,93). Finally, intrinsic characteristics of apheresis products may be another limitation factor for the generation of autologous CAR T-cells (94). In the myeloma setting, harvesting the sufficient number of Tcells from patients seems to be feasible to produce CAR Tcells [e.g., the bb2121 clinical trial has shown that in 100% of patients who underwent leukapheresis, BCMA CAR T-cells were successfully generated (31)].…”

Group O plasma as a media supplement for CAR‐T cells and other adoptive T‐cell therapies

“…The RBC concentration of the starting materials has previously been shown to affect the final phenotype of CAR-T products (specifically increasing the CD4:CD8 ratio). 33 In this study, we found that presence of contaminating RBCs in lymphocyte culture was independently associated with reduced lymphocyte expansion and phenotypic alteration of lymphocytes to a less desirable (increased percentage of T EM ) phenotype. 34,35 Additionally, significant differences in transcriptional activity and protein expression were seen in cultures with contaminating RBCs.…”