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.
Bibliographical noteFunding Information:
Support was provided by the UCLA Depart ment of Pediatrics 3K12HD034610 and the NIH/ NHLBI 2T32HL086345-06 Training Grant in Developmental Hematology, NCI 1K23CA222659, American Society of Hematology Scholar Award, Hyundai Hope on Wheels Scholar Award, Miranda D. Beck Pediatric Cancer Research Foundation, Gwynne Hazen Cherry Memorial Fund, the Pediatric Cancer Research Fund, UCLA Children’s Discovery and Innovation Institute, UCLA Jonsson Comprehensive Cancer Center, UCLA Cancer Research Coordinating Committee, UCLA Clinical and Translational Science Institute Grant UL1TR000124, and the St. Baldrick’s Foundation. All the authors acknowledge the collaboration with Drs. Stephen Forman and Christine Brown, from City of Hope, who provided the EGFRt and EQ CAR constructs, and the training and technical support of the Flow Cytometry Core of the UCLA Broad Stem Cell Research Center (BSCRC), where all flow cytometry experiments were performed. The authors also acknowledge the valuable scientific advice and technical support from Dr. Gay Crooks, MBBS, and Donald B. Kohn, MD, and the help on data acquisition and analyses from Felix Bergara Salazar, Andy Tu, TulikaTyagi,Therese Dinoso,and NeziaRahman.
© Copyright 2019, Mary Ann Liebert, Inc., publishers 2019.
- HSC, CAR, cancer immunotherapy, EGFR, cetuximab, gene therapy