Academic manufacturing of anti-CD19 CAR-T cells from a leukapheresis with a high proportion of acute myeloid leukaemia blasts.

Abstract

This report describes the manufacturing of anti-CD19 chimeric antigen receptor-T (CAR-T) cells in an academic setting for a patient with a high tumour burden (>90%) who was referred for CD19+ acute myeloid leukaemia (AML). A 30-year-old woman with refractory acute myeloid leukaemia (AML) was found with 94% circulating blasts, 79% of which were considered CD19 positive upon initial screening. She was selected as a candidate for inclusion in an anti-CD19 CAR-T cell clinical trial. In order to promptly offer a therapeutic option to this patient with active disease, an apheresis product was magnetically enriched to >90% T cells, enabling manufacturing of a clinically compliant CAR-T cell dose (> 1 × 106 CAR-T cells/kg). A potency assay confirmed CD19-specific cytotoxic activity. Yet, further in-process flow cytometry quality controls unexpectedly revealed that the AML blasts did not actually express CD19, which was confirmed by reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), eventually rendering the patient ineligible for treatment. This case demonstrates the feasibility of manufacturing functional CAR-T cells from an apheresis product containing less than 3% T cells and indicates that a second target validation should be considered prior to recruitment when targeting a lymphoid marker in rare cases of myeloid leukaemia.