Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells

David F Stroncek,Jiaqiang Ren,Hanh Khuu,Steven Highfill,Crystal L Mackall,Marianna Sabatino,Rosandra N Kaplan,Nirali N Shah,Terry J Fry,Daniel W Lee

doi:10.1186/s12967-017-1160-5

Abstract

BackgroundClinical 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.MethodsCounter-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.ResultsWhen 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.ConclusionsEnrichment 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.

Highlights

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
We report the results of manufacturing CD19- and GD2-CAR T cells using lymphocytes collected by apheresis and enriched by elutriation as starting material
Composition of the PBMC concentrates and elutriated lymphocytes We manufactured 8 CD19 CAR T cell products and 5 GD2-CAR T cell products using autologous PBMC concentrates collected by apheresis and enriched for lymphocytes by elutriation

Summary

Introduction

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. Most CAR T cell manufacturing protocols initate cell production with autologous T cells collected by apheresis using a blood cell separator which separates lymphocytes from plasma, platelets, red blood cells (RBCs) and granulocytes. The lymphocyte-rich peripheral blood mononuclear cell (PBMC) concentrates collected by apheresis are enriched for monocytes and contain variable quantities of RBCs, platelets and granulocytes.

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