Development and characterisation of NKp44‐based chimeric antigen receptors that confer T cells with NK cell‐like specificity

Objectives One of the reasons as to why chimeric antigen receptors (CAR)‐T cell therapy for malignancies other than CD19‐ or BCMA‐positive tumors has yet to produce remarkable progress is the paucity of targetable antigens. NKp44 is only expressed by activated natural killer cells and detects a variety of transformed cells, while it reportedly does not react with normal tissues. The aim of this study is to develop CAR‐T cell that can target multiple types of tumor cells. Methods We created a series of novel CAR constructs in first‐generation (1G) and second‐generation (2G) CAR format with the extracellular immunoglobulin‐like domain of NKp44 (NKp44‐CAR). Results Transduction of the best 1G construct into human primary T cells led to specific cytotoxic effects and cytokine secretion upon encountering multiple types of neoplastic cells including AML, T‐ALL and childhood solid tumors. Replacement of the extracellular hinge domain of NKp44 with that of CD8α resulted in diminished CAR function. The 1G NKp44‐CAR‐T cells exhibited significantly better tumor control in long‐term co‐culture assays compared with activated NK cells, as well as with NK cells transduced with identical NKp44‐CAR. T cells transduced with the best 2G‐CAR construct with 4‐1BB co‐stimulatory domain proliferated at significantly higher levels upon single antigen exposure and showed significantly better tumor control compared with the 1G‐CAR and 2G‐CAR with CD28 co‐stimulatory domain. Conclusions NKp44‐based CAR endows T cells with NK cell‐like anti‐tumor specificity. The CAR gene created in this study will be useful for the development of novel gene‐modified T‐cell immunotherapy. A challenge in the development of effective chimeric antigen receptor transduced T (CAR‐T) cell therapy is to identify an appropriate target on tumor cells. We created a series of novel CAR constructs with the extracellular immunoglobulin‐like domain of NKp44, one of the activating receptors expressed on activated natural killer cells. Human T cells transduced with optimised NKp44‐based CAR constructs exerted specific cytotoxic effects and cytokine secretion upon encountering multiple types of neoplastic cells, including those associated with acute myeloid leukaemia, T‐cell acute lymphoblastic leukaemia, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, neuroblastoma and glioblastoma cells.