Development of allogenic nonviral RNA-based CAR-NK therapy targeting CDH17 in relapsed/refractory gastrointestinal cancer.

e14539 Background: Gastrointestinal (GI) cancers are commonly fatal after metastasis. The incidence and mortality of GI tumors are high, and the benefits of existing therapies are limited. Cadherin-17 (CDH17) is highly prevalent and specific to GI cancers which has potential for the development of novel treatments for GI cancers. Cell-based immunotherapy using genetically engineered autologous Chimeric Antigen Receptors T (CAR-T) cells has been clinically efficacious for treating hematological malignancies. However, its manufacturing is extremely expensive and lengthy with ~10% failure production rate. Equipping NK cells with CAR may represent a safer and equally effective approach for use in solid tumors as an alternative to autologous CAR-T-cell therapy. Nonviral RNA-based expression of CAR vector in NK cells will provide immediate and multiple accessibility to cancer patients, lower risk of abnormal genetic alteration mediated by viral integration, and potentially limit side effects. This paper demonstrates the development of RNA-CAR-NK targeting CDH17-positive GI cancers. Methods: Ex vivo expanded human peripheral blood NK (Magicell-NK, Medigen, Taiwan) cells were used in this study. To generate anti-CDH17 (C7) CAR-NK cells, mRNA containing a novel CDH17-specific single-chain variable fragment and the 4-1BB costimulatory domain was electroporated to Magicell-NK cell. Flow cytometry analysis was performed to evaluate the cell viability and the expression level of CAR and cytotoxic surface markers of the CAR-NK cells. CDH17 specific cytolytic activity against AGS gastric cancer cell line and the concentration of cytokines released by CAR-NK cells were measured by in vitro cytotoxicity assay and cytokine release assay, respectively. Results: Our results demonstrated efficient electroporation with high-level C7 CAR expression ( > 80%) and optimal CAR-NK cell viability (~60%). Interestingly, 12 h co-culture of C7 CAR-NK cells with AGS gastric cancer cells at 1:1 and 2:1 effector/target (E:T) ratio showed a significantly enhanced antitumor response of C7 CAR-NK cells on AGS-CDH17+ cells but not on AGS-CDH17- cells, indicating CDH17 target-specific cytolytic activity. Moreover, we observed an elevated level of cytokines such as INF-γ and TNF-α released by C7 CAR-NK cells only in co-culture with AGS-CDH17+ cells, confirming the mechanisms behind the CAR-mediated cytotoxicity. Furthermore, these results were supported by an increased level of surface CD107a or LAMP-1 expression in C7 CAR-NK cells during the 12 h of co-culture with AGS-CDH17+ cells at 1:1 and 2:1 E:T ratio. Conclusions: Our findings suggest that a non-viral based CDH17 CAR-NK cell therapy could be a potential allogeneic, off-the-shelf CAR-NK therapy candidate in GI cancer and support the rationale of further investigating it in vivo and clinical trials.