EXTH-02. A TERT-SPECIFIC TCR-LIKE ANTIBODY CAR T CELL APPROACH FACILITATES KILLING OF GLIOBLASTOMA

Abstract

Abstract Glioblastoma (GBM) is a difficult form of brain cancer to treat as GBM cells utilize various strategies to evade host immune responses. Prior studies have shown autologous cytotoxic T cells (CTL) modified to express chimeric antigen receptors (CAR) facilitating host CTL responses against cancers. This study investigates the ability of T-cell receptor (TCR)-like antibody clones against TERT to facilitate specific CAR T cell mediated killing of GBM. Lentiviral vectors were created for five TCR-like antibody CAR constructs recognizing an HLA-A*02:01-restricted TERT-derived antigen. Nur77-GFP Jurkat reporter cell lines and 3 healthy donor-derived CTLs transduced with these vectors were used in flow-based and in vitro killing assays to assess specificity and functionality. K562 cells that did, and did not, express HLA-A*02:01 were targets in the flow-based assay. Flow cytometry captured activation of transduced Jurkat cells via GFP and CD69 upregulation. Three luciferase-transduced GBM cell lines were targets in the cytotoxic killing assay and visualized via bioluminescence imaging (BLI). Increased GFP and CD69 expression was seen on Jurkat cells transduced with clones 3A12 and 3H2 only when co-cultured with TERT antigen and A*02 positive K562 suggesting specific recognition of the TERT:HLA complex. Furthermore, healthy donor-derived CTL transduced with 3A12 and 3H2 CAR constructs demonstrated in vitro killing of tumor cells by BLI. Interestingly, the 3H2 construct only showed cytotoxicity in the presence of exogenous TERT antigen. However, when the heavy and light chains of the 3H2 clone were flipped in the lentiviral construct (3H2F), specificity to the TERT:HLA complex was preserved but the CAR T cells now demonstrated cytotoxicity of GBM without needing exogeneous peptide. TCR-like antibody clones 3A12 and 3H2F specifically recognize an HLA-A*02:01-restricted TERT-derived antigen, facilitating effective in vitro cytotoxicity of GBM cells. Ongoing experiments will evaluate the in vivo activity of these TERT-specific CAR constructs and assess potential off-target toxicity.