Kasahara et al. transduced primary human T and NK cells with a series of CARs comprised of the NKp44 or NKp30 extracellular ligand binding domains and various hinge and endodomain components. The combinations greatly impacted CAR cell surface expression, in vitro IFNγ induction, and cytotoxic effects against various tumor cell lines expressing specific ligands. NKp44-CAR T cells survived longer and mediated greater serial killing of tumor cells than CAR-NK or ex vivo-activated NK cells. The optimal CAR comprised the NKp44 ligand binding domain, CD8α hinge region, CD8α transmembrane, and 4-1BB and CD3ζ signalling domains.
Contributed by Paula Hochman
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 CD8alpha 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.