Genetic engineered primary human natural killer cells targeting TIGIT via CRISPR/Cas9 enhanced anti-tumor activity against GBM spheroids and 2D culture.

Background. Although several immunotherapies against glioblastoma (GBM) have been investigated for long time, only limited effective results are acquired. Therefore, we developed immunotherapy based on genome edited NK cells knocking out the checkpoint receptor, which would overcome the immunosuppressive tumor microenvironment in GBM. Methods. We generated T cell immunoglobulin and ITIM domain (TIGIT), an inhibitory receptor expressed on lymphocytes, knockout (KO) human primary NK cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein9 (Cas9), each single guide RNA targeting different genome sites on TIGIT coding exons. To detect anti-tumor activity of genome edited NK cells against GBM, we utilized 2D adherent model and spheroids derived from GBM cell lines, U87, T98G, LN18, and U251. Subsequently, we performed real time cell growth assays, flow cytometry based apoptosis assays, and ELISA for investigating anti-tumor activity. Result. We established TIGIT KO human primary NK cells using CRISPR/Cas9. Flow cytometry indicated effective knockout of TIGIT and unchanged expression of immune checkpoint receptors other than TIGIT. T7 endonuclease I mutation detection assays showed that RNPs disrupted the intended genome sites. Using real time cell growth assays, we revealed enhanced anti-tumor activity of genome edited NK cells against 2D adherent GBM cells. The genome edited NK cells also exhibits enhanced anti-tumor effect against GBM spheroids derived from GBM cell lines.Conclusion. Our founding suggests that immunotherapy based on TIGIT KO NK cells using CRISPR/Cas9 is a promising therapy against GBM. Despite multimodality treatment, patients with glioblastoma (GBM) exhibits poor outcome. A novel strategy is needed and the immunotherapy is one of them. Although immunotherapies like chimeric antigen receptors (CAR) T therapy, immune checkpoint inhibitors, vaccine therapy and oncolytic virus therapy are investigated, it is difficult to change the severe immunosuppressive tumor microenvironment, so called “cold tumor”. To activate cancer immunity cycle, NK cells, which recognize tumor cells via several receptors and promote each step of cancer immunity cycle, are the suitable agents for immunotherapy against GBM. Furthermore, Genome edited NK cells targeting TIGIT, an immune checkpoint receptor, have an advantage in that be able to maintain brockage of TIGIT signal in result avoiding exhausted status, whereas the effect of anti TIGIT antibody depends on its dose and administration timing. Here, we established TIGIT KO human primary NK cells and demonstrated enhanced anti-tumor activity. This designer NK cells will thus provide more insight into the GBM treatment.