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A Cetuximab-Mediated Suicide System in Chimeric Antigen Receptor–Modified Hematopoietic Stem Cells for Cancer Therapy

  • Kao, Roy L.1
  • Truscott, Laurel C.1
  • Chiou, Tzu-Ting1
  • Tsai, Wenting2
  • Wu, Anna M.2
  • De Oliveira, Satiro N.1
  • 1 Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, California
  • 2 Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California.
Published Article
Human Gene Therapy
Mary Ann Liebert
Publication Date
Apr 01, 2019
DOI: 10.1089/hum.2018.180
PMID: 30860401
PMCID: PMC6479239
PubMed Central


Using gene modification of hematopoietic stem cells (HSC) to create persistent generation of multilineage immune effectors to target cancer cells directly is proposed. Gene-modified human 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. Truncated epidermal growth factor receptor (EGFRt) was tested as a suicide gene system co-delivered with anti-CD19 chimeric antigen receptor (CAR) to human HSC. 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. An antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used, incubating target cells with leukocytes and monoclonal antibody cetuximab to determine the 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, droplet digital polymerase chain reaction (ddPCR), and positron emission tomography (PET) imaging using 89Zr-Cetuximab. 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: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. These results give proof of principle for CAR-modified HSC regulated by a 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.

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