Hematopoietic stem cell (HSC) transplantation is a beneficial therapeutic option for malignant and monogenic diseases. The current clinical approach to bone marrow conditioning involves high-dose chemotherapy and/or whole-body irradiation, but because of the high toxicity has low applicability. In this project we test an alternative approach to myeloablation utilizing Adapter CAR™ T (AdCAR-T) cells and aim to determine whether it is capable of effective myeloablation in vitro.

We optimized the cytotoxicity of the AdCAR-T cell system towards HSCs in vitro using flow cytometry, colony-forming unit (CFU) assays and MACSplex cytokine assays. Our optimized assay conditions include an effector to target cell ratio of 10:1, 10 µg/ml of adapter in TexMACS Medium™ and incubation for at least 24h. AdCAR-T cell functionality in these conditions was demonstrated by the secretion of specific cytokines such as GM-CSF, TNFa, and IFNγ.

In addition, adapters against a broad range of HSC surface antigens such as CD34, CD33, CD117 and CD135 were tested. Of all the adapters, the anti-CD34 exhibited the highest HSC lysis, ranging from 40% to 90%. CFU assays corroborated these results, with a decrease of colony count that often exceeded the level of cytotoxicity observed by flow cytometry. At least 10% of HSCs appear to resist lysis under all assay conditions, and these cells are currently under investigation using the MACSima Imaging Platform and NGS-based transcriptome analysis. Future experiments in mice will indicate if the HSC lysis observed in vitro can be successfully translated into complete myeloablation in vivo.