Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells

Abstract

Background: Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes.

Methods: Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products.

Results: When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells.

Conclusions: Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

Keywords: Cancer immunotherapy; Cellular therapy; Chimeric antigen receptor T cells; Elutriation; Myeloid derived suppressor cells; Peripheral blood mononuclear cells; T cells.

Fig. 1

Quantity of T cells and transduced T cells in GD2-CAR T cell products. GD2-CAR T cells were manufactured over 10 or 11 days using autologous PBMC concentrates enriched for lymphocytes using one of 3 methods: anti-CD3/CD28 bead selection (n = 6, circles), anti-CD3/CD28 bead selection plus plastic adherence (n = 5, triangle) and elutriation (n = 5, diamonds). The quantity of CD3 + cells in the final product is shown in a and transduced CD3+ cells in b

Fig. 2

Quantity of T cells and transduced T cells in CD19-CAR T cell products. CD19-CAR T cells were manufactured over 7–9 days using autologous PBMC concentrates enriched for lymphocytes using one of 3 methods: anti-CD3/CD28 bead selection (n = 6, circles), anti-CD3/CD28 bead selection plus plastic adherence (n = 16, triangle) and elutriation (n = 8, diamonds). The quantity of CD3+ cells in the final product is shown in a and transduced CD3+ cells in b