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 CD8 alpha 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.