靶向 IL13Rα2 阳性人胶质瘤的新型 CAR-T 细胞的体外和体内鉴定与表征

Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13R 2 fragment fused with CD3 and co-stimulatory cytoplasmic domains of CD28 and 4-1BB.

We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13R 2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells.

The binding of 14-1 clone is to IL-13R 2Fc-chimeric protein is 5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13R 2Fc and cell free supernatants only from IL-13R 2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13R 2-CAR-T cells specifically killed IL-13R 2+ve but not IL-13R 2-ve tumour cells in vitro and selectively caused significant release of IFN- only from IL-13R 2+ve co-cultures. These CAR-T cells regressed IL-13R 2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13R 2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy.

Taken together, we conclude that the novel scFv-IL-13R 2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.