A cetuximab-mediated suicide system in CAR-modified hematopoietic stem cells for cancer therapy.

BACKGROUND: We propose using gene-modification of HSC to create persistent generation of multilineage immune effectors to directly target cancer cells. Gene-modified human hematopoietic stem cells (HSC) have been used to introduce genes to correct, prevent or treat diseases. Concerns regarding malignant transformation, abnormal hematopoiesis and autoimmunity exist, making the co-delivery of a suicide gene a necessary safety measure. We tested truncated epidermal growth factor receptor (EGFRt) as a suicide gene system co-delivered with anti-CD19 chimeric antigen receptor (CAR) to human HSC.

METHODS: Third-generation self-inactivating lentiviral vectors were used to co-deliver an anti-CD19 CAR and EGFRt. In vitro, gene-modified HSC were differentiated into myeloid cells to allow transgene expression. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used incubating target cells with leukocytes and monoclonal antibody cetuximab to determine percentage of surviving cells. In vivo, gene-modified HSC were engrafted into NSG mice with subsequent treatment with intraperitoneal cetuximab. Persistence of gene-modified cells was assessed by flow cytometry, ddPCR, and PET imaging using 89Zr-Cetuximab.

RESULTS: Cytotoxicity was significantly increased (p=0.01) in target cells expressing EGFRt after incubation with leukocytes and cetuximab 1 µg/mL, compared to EGFRt+ cells without cetuximab, and non-transduced cells with or without cetuximab, at all effector-to-target ratios. Mice humanized with gene-modified HSC presented significant ablation of gene-modified cells after treatment (p=0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues. PET imaging confirmed ablation with a decrease of an average of 82.5% after cetuximab treatment.

CONCLUSIONS: These results give proof of principle for CAR-modified HSC regulated by suicide gene; further studies are needed to enable clinical translation. Cetuximab ADCC of EGFRt-modified cells caused effective killing. Different ablation approaches, such as inducible caspase 9 or co-delivery of other inert cell markers should also be evaluated.