Allogeneic stem cell transplantation is a curative therapy for refractory, relapsed and high risk leukaemia and lymphoma. Despite improvements in leukaemia treatment the disease relapse occurs in 30-50% of patients and remains unacceptably high. There is thus a clear need for novel agents in the treatment of leukaemia relapse. Leukaemia specific T cells genetically modified with Chimeric Antigen Receptor (CAR) generated in the laboratory to target malignant cells is a novel approach with proven success in early phase human trials. Patient's own T cells can be genetically modified in the laboratory to target tumour associated antigens through the introduction of CAR. CAR-modified T cells are amplified ex vivo to numbers suitable for adoptive cell therapy and administered to the patient upon preconditioning. Unfortunately, CAR-modified mature T cells often rapidly differentiate into short-lived effector cells that exhibit limited anti-tumour activity in vivo.
Here we propose to use CAR-modified haematopoietic stem cells (HSC) to generate CART- cells. We hypothesise that CAR-modified HSC will provide long-lasting supply of CAR-T cells mediating sustained anti-tumour activity. Umbilical cord blood-derived CD34+ cells enriched with HSC were genetically modified to express CAR targeting CD19 antigen(CAR19) widely expressed on B-cell malignancies. An optimised gamma-retroviral gene transfer method was used to transduce CD34+ stem cells with CAR19. CAR19-transduced stem cells generate CD33+ myeloid, CD56+ natural killer and CD34-CD7+ T cell precursors expressing CAR19 and undergo normal proliferation and differentiation in vitro in colony forming unit (CFU) assay. B cell development analysed in co-cultures with bone marrow stroma MS5 cells, however, was completely suppressed.
CAR19-transduced CD34+ stem cells were transplanted to immunocompromised NOD-SCID IL2Rg-/- (NSG) mice to generate multilineage immune effector cells exhibited delayed early engraftment. It is relevant that CD19+ B cells regenerate first in this mouse model. Thus mice transplanted with CAR19-transduced stem cells exhibited severe CD19+ B cell depletion confirming the functionality of CAR19 expressed in human B cells. To promote T cell regeneration, CD34+ HSCs were co-cultured with Notch ligand DL4 for 1 week before infusion. CD3+T cells were detectable in the blood of mice infused with DL4-pre-cultured CD34+ HSCs as soon as 6 weeks post-transplant while control CD34+ stem cells that were not co-cultured without DL4 exhibited significantly delayed T cell regeneration. Importantly, reduced leukaemia burden was observed in mice reconstituted with CAR19-modified stem cells and infused with human CD19+ leukaemia NALM6 cells. These data proves the feasibility of the stem cell approach to generate potent immune effector cells capable of fighting cancer. Further analysis aimed to identify the role of different multilineage immune effector cells in mediating anti-tumour activity in stem cell reconstituted mice is currently being conducted.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.
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