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

Abstract

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, genemodified HSC were differentiated into myeloid cells to allow transgene expression. An antibodydependent 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 89 Zr-Cetuximab. Cytotoxicity was significantly increased ( p = 0.01) in target cells expressing EGFRt after incubation with leukocytes and cetuximab 1 lg/ 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 genemodified 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.

Bispecific T-Cell Redirection versus Chimeric Antigen Receptor (CAR)-T Cells as Approaches to Kill Cancer Cells

“…As mentioned in Section 6.3 (see also Figure 9), the RQR8 epitope was constructed as a target for the anti-CD20 mAb rituximab [302] and has been employed in allogeneic cells as a cell-surface kill switch [301,303]. Additionally, a truncated version of epidermal growth factor receptor (EGFRt), which encompasses residues 334–668 of mature EGFR (domains III and IV), has been fused to the GM-CSF leader peptide to make a surface expressed “tag” for CAR-T cells [348,349,350]. This EGFRt tag is recognized by the approved drug cetuximab (Erbitux ® ), which can then be used as a kill switch to eliminate cells expressing the tag [348,349,350].…”

“…Additionally, a truncated version of epidermal growth factor receptor (EGFRt), which encompasses residues 334–668 of mature EGFR (domains III and IV), has been fused to the GM-CSF leader peptide to make a surface expressed “tag” for CAR-T cells [348,349,350]. This EGFRt tag is recognized by the approved drug cetuximab (Erbitux ® ), which can then be used as a kill switch to eliminate cells expressing the tag [348,349,350]. There are currently at least 12 unique CAR-T cell constructs in clinical trials that have incorporated the EGFRt tag (WR Strohl, BiStro Biotech Consulting Antibody and CAR-T Database, last updated 20 June 2019).…”

Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration

“…Epidermal growth factor receptor (EGFR) is not expressed by cells of the hematopoietic and lymphopoietic systems, which makes it as an attractive therapeutic molecule 63 . The truncated EGFR (EGFRt) could substitute CD90 as a selection marker, as well as a similar suicide gene [63][64][65] . The α-EGFR mAb, cetuximab, which has been widely used in clinical practice, could be used to easily eliminate the engineered LLPCs with EGFRt expression on the surface in vivo to avoid possible adverse effects.…”

CARs: Beyond T Cells and T Cell-Derived Signaling Domains

“…While this solves the issue of cell exhaustion faced in conventional CAR-T cell therapy, it also calls for reliable safety switches to control toxicity and the risk of malignant transformation [76]. An example for a safety switch gene successfully tested by Kao et al is the truncated epidermal growth factor receptor (EGFRt), mediating ADCC [79]. The presence of many different CAR effector cell types at the same time promises multi-level anti-tumor activity, employing all arms of the immune system [77].…”

“…To demonstrate the contribution of non-T cells to the cytotoxicity, they repeated the experiment in SCID mice (that are incapable generating mature T cells) [80]. Several other groups also proved HSC-derived CAR myeloid cells to significantly contribute to cytotoxicity of this multi-lineage approach [79,81,82]. Of note, most studies working on CAR-transduced HSCs do not address myeloid cells, but focus more on the potential of a self-renewing CAR-T cell response [78].…”