A Novel Peptide-MHC Targeted Chimeric Antigen Receptor T Cell Forms a T Cell-like Immune Synapse

Chimeric Antigen Receptor (CAR) T cell therapy is a promising form of adoptive cell therapy that re-engineers patient-derived T cells to express a hybrid receptor specific to a tumour-specific antigen of choice. Many well-characterised tumour antigens are intracellular and therefore not accessible to antibodies at the cell surface. Therefore, the ability to target peptide-MHC tumour targets with antibodies is key for wider applicability of CAR T cell therapy in cancer. One way to evaluate the effectiveness and efficiency of ligating tumour target cells is studying the immune synapse. Here we generated a second-generation CAR to targeting the HLA-A*02:01 restricted H3.3K27M epitope, identified as a possible therapeutic target in ~75% of diffuse midline gliomas, used as a model antigen to study the immune synapse. The pMHCI-specific CAR demonstrated specificity, potent activation, cytokine secretion and cytotoxic function. Furthermore, we characterised killing kinetics using live cell imaging as well as CAR synapse confocal imaging. Here we provide evidence of robust CAR targeting of a model peptide-MHC antigen and that, in contrast to protein-specific CARs, these CARs form a TCR-like immune synapse which facilitates TCR-like killing kinetics.