In Molecular Therapy: EBAG9 silencing exerts immune checkpoint function without adverse effects

Wirges A, Bunse M, Joedicke JJ, Blanc E, Gudipati V, Moles MW, Shiku H, Beule D, Huppa JB, Höpken UE, Rehm A

Read in Molecular Therapy

Chimeric antigen receptor (CAR) T cells have demonstrated clinical efficacy against a number of hematologic malignancies. Despite significant response rates and durable remissions, a large number of patients relapse. Treatment options post-CAR relapse are limited, resulting in challenges to producing a therapy with a complete response for these cancers. Cytolytic CD8+ T cells (CTLs) have been engineered to meet the need for more efficacious CAR T-cell therapies. These cells demonstrate anti-tumor activity by secreting effector molecules such as cytokines and cytotoxic granules. One strategy to improve the potency of CTLs aims to enhance the intracellular pathways responsible for transport and secretion of the effector molecules. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) negatively regulates secretion of effector molecules. Previous studies have shown that deletion of the Ebag9 gene in mice results in enhanced release of lytic granules from secretory lysosomes in CD8+ T cells. In this study, Wirges et al demonstrate that EBAG9 downregulation in murine T cells and human CAR T cells augmented the cytolytic activity of these cells against hematopoietic tumors in vitro and in vivo.

Previously, this group had identified the inhibitory effect of EBAG9 on the cytotoxic activity of T cells. To take advantage of this effect, they engineered a retroviral vector encoding GFP to express miRNAs targeting the mouse Ebag9 reading frame. After transduction with this vector in murine primary T cells, significant reduction of Ebag9 expression was observed for two of the miRNAs (miR-M1 and miR-M2) at the mRNA and protein levels. The in vivo cytotoxicity of the miRNA-modified CTLs was assessed by transducing T cells isolated from mice immunized with SV40-large T antigen (TAg) with the miR-M1 retroviral vector. GFP+ transduced cells were sorted and transplanted into immunodeficient mice also immunized with TAg and later challenged with TAg peptide-loaded splenocytes; recipients that had received the miR-M1 vector versus controls demonstrated a two-fold higher antigen specific killing rate.

To assess this strategy in a human setting, retroviral miRNA vectors targeting the human EBAG9 gene were generated and tested for their ability to achieve EBAG9 mRNA and protein knockdown in Jurkat T cells. For one of the miRNAs demonstrating activity (miR-H18), the GFP transgene of the retroviral vectors was exchanged for a B cell maturation antigen (BCMA) CAR T cell, effective against multiple myeloma. The miR-H18 BCMA CARs released higher amounts of the effector molecule granzyme A than controls in transduction experiments, and were specifically activated when cultured in the presence of multiple myeloma cells. Further killing assays identified that miRNA-mediated silencing of EBAG9 in BCMA CAR T cells resulted in more potent cytotoxicity of the CARs in BCMA+ myltiple myeloma and B cell non-Hodgkin lymphoma cell lines. The kinetics of the improved cell killing with the miR-H18 BCMA CAR T cell were characterized by microscopy and found to be universally applicable for increasing the potency of CD8+ CTLs. Studies performed in an in vitro serial transfer model demonstrated that the BCMA CAR and miRNA-edited BCMA CAR T cells maintained their effector and proliferation capacities after multiple rounds of stimulation without evidence of exhaustion or activation-induced cell death. Transcriptome profiling of the miR-H18 mediated EBAG9  silencing ultimately showed that exhaustion-related genes were downregulated, indicating that increased cytotoxic potency did not impair T-cell fitness. Finally, treatment with the BCMA CAR T cells in a xenotransplantation model showed potent suppression of tumor growth without demonstration of CAR T-cell exhaustion. In summary, the results demonstrate the improved therapeutic potency of EBAG9-silenced T cells, suggesting their clinical application as a promising option for cancer immunotherapy.

Dr. Schneller is an Associate Member of ASGCT and Junior Editor of The Vector.  

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