NKG2D-TIR CAR-Macrophage: A Novel Anti-Tumour Strategy

Abstract

Background: When compared to the conventional CAR-T therapies, Chimeric Antigen Receptor (CAR) macrophages represent a promising strategy to target solid tumours. To enhance therapeutic outcome, variants have been developed, a notable example is the NKG2D CAR M, which targets the diverse NKG2D ligands expressed in cancer cells, offering a broad spectrum ability that recognizes various antigens in stressed cancer cells. This study aims to determine whether the incorporation of the TIR signalling domain into NKG2D-based chimeric antigen receptor enhances mediated anti-tumour immunity. Method: To achieve this, molecular cloning techniques were used to generate a CAR construct, which was integrated into a lentiviral packaging system to facilitate gene delivery. Restriction digest, colony PCR, and sequence alignment were used to confirm the structural integrity of the construct, and flow cytometry was used to validate the efficiency of transduction via reporter gene expression. However, the construct was remodified through mCherry substitution with enhanced green fluorescent protein (EGFP) to improve the downstream confirmation of NKG2D expression using fluorescence-associated cell sorting (FACS). Result: Preliminary results confirmed successful expression of the CAR fluorescence reporter, achieved using flow cytometry. Sequencing data also showed aligned functional CAR. Conclusion: Although expression and functional validation were not assessed within the scope of this study, the successful engineering and preparation of the CAR construct establish a fundamental framework for advance therapeutic testing.