Background: Gene-modified human hematopoietic stem cells (HSC) have been used to introduce genes to correct, prevent or treat diseases. Clinical trials using infusion of autologous T cells engineered with anti-CD19 chimeric antigen receptors (CAR) have demonstrated complete remissions even in chemotherapy-resistant malignancies, but the persistence of the effector cells is transient, limiting clinical efficacy and allowing the cancer to recur. Current trials of CAR T cells have recommended patients in remission post-T cell therapy to undergo hematopoietic stem cell transplantation (HSCT) due to the limited persistence of the CAR+ T cells. Seeking to foster persistence of the CAR-modified immune cells, we propose using gene modification of HSC to create persistent generation of multilineage immune effectors to directly target cancer cells. Concerns regarding malignant transformation, abnormal hematopoiesis and autoimmunity exist, making the co-delivery of a suicide gene a necessary safety measure. We evaluated a second-generation anti-CD19 chimeric antigen receptor (CAR) to human HSC in a humanized mouse model, co-delivered with a truncated epidermal growth factor receptor (EGFRt) as a suicide gene system.

Significance: CAR+ HSC can be infused in the context of HSCT, when the chemotherapy-conditioning regimen favors engraftment of gene-modified cells, decreases residual tumor burden and decreases the chance of CAR immunogenicity. The prospect of modifying autologous cells to enhance GVL bears the possibility of decreased morbidity and mortality, a concern for specifically vulnerable populations such as children and elderly patients. In addition, this approach will offer alternative therapy for those without cell sources available for allogeneic HSCT, benefiting ethnic minorities.

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 immunodeficient NSG mice treated or not with intraperitoneal cetuximab 1mg/mouse/day for 12 days. Persistence of gene-modified cells was assessed by flow cytometry, ddPCR, and PET imaging using 89Zr-Cetuximab.

Results: Gene modification of HSC did not affect engraftment, proliferation or differentiation, at efficiency of 20-40%. Antigen specificity of myeloid, NK and T cells were successfully redirected against CD19. Ablation of gene-modified cells was significantly increased (p=0.01) in target cells expressing EGFRt after incubation with leukocytes and cetuximab 1µg/mL, compared to EGFRt+ cells without cetuximab, and non-transduced cells with or without cetuximab. This was seen at all effector-to-target ratios. Mice engrafted with CAR-modified HSC presented prolonged persistence of gene-modified cells in bone marrow, spleen, thymus and peripheral blood, and CAR+ cells elicited protection against CD19+ tumors, with inhibition or elimination of tumor development and survival advantage. Mice humanized with gene-modified HSC for the expression of the suicide gene had significant ablation of gene-modified cells after treatment with cetuximab (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 of humanized mice documented engraftment of EGFRt-expressing cells in bone marrow, spleen and liver, and confirmed ablation with an average decrease of 82.5% after cetuximab treatment.

Conclusions: These results give proof of principle for persistence and anti-tumor activity of CAR-modified HSC regulated by suicide gene, and 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 be evaluated.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.