LMP2A-Targeting CAR-T Cells Equipped With Inducible IL-18 to Address EBV-Associated Malignancies

Epstein-Barr virus (EBV) infects up to 95% of the world's population and persists in B cells and epithelial cells. Uncontrolled proliferation of EBV-infected cells can result in the development of EBV-associated malignancies, for example, post-transplant lymphoproliferative disorder (PTLD) or nasopharyngeal cancer (NPC). It is estimated that 1.8% of deaths due to cancer worldwide are associated with EBV, and the treatment options are limited. As a new therapeutic approach, we developed chimeric antigen receptor T cells targeting EBV-derived latent membrane protein 2A (LMP2A_CAR-Ts). To enable specific elimination of malignant B cells infected with the intracellular pathogen, we utilised T-cell receptor (TCR)-like specificity to generate a CAR against the LMP2A-derived peptide CLGGLLTMV (CLG) presented in the context of HLA-A*02:01. To increase functionality in the tumour microenvironment, LMP2A_CAR-Ts were additionally equipped with inducible release of IL-12 (LMP2A_iIL-12_TRUCKs) or IL-18 (LMP2A_iIL-18_TRUCKs). LMP2A_CAR-Ts and LMP2A_iIL-18_TRUCKs specifically recognised HLA-A*02:01⁺ EBV-transformed B-lymphoblastoid cell lines and HLA-A*02:01⁺ cells loaded with CLG peptide (A02_CLG⁺ cells), proving their target specificity, while, unexpectedly, LMP2A_iIL-12_TRUCKs exhibited a disparate T_EM and NK-like phenotype and A02_CLG-independent reactivity. In contrast, LMP2A_CAR-Ts and LMP2A_iIL-18_TRUCKs effectively induced T-cell signalling, activation, release of cytotoxic mediators, including IL-18 by LMP2A_iIL-18_TRUCKs and mediated cytotoxicity in a target-specific manner. Our results demonstrate that LMP2A_CAR-Ts and LMP2A_iIL-18_TRUCKs specifically recognise the EBV-derived CLG peptide presented in the context of HLA-A*02:01. Especially, LMP2A_iIL-18_TRUCKs with an even improved anti-tumour response, as well as the potential to recruit bystander immune cells and overcome EBV-mediated immune evasion strategies, might serve as a novel treatment option for various EBV-associated malignancies.