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

Abstract

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.

NKp44-based chimeric antigen receptor effectively redirects primary T cells against synovial sarcoma

“…The MSCV-IRES-GFP vector, pEQ-PAM3(-E), and pRDF plasmids were obtained from St. Jude Vector Development and Production Shared Resource. Various NKp44-based CAR genes with structural and functional diversity have been developed in our laboratory [17] . The CD19-targeted 4-1BB co-stimulated CAR gene has been reported previously [18] .…”

“…NKp44 (also known as NCR2) is an NCR that binds to several ligands on the surface of tumor cells or virally infected cells, including a peculiar isoform of mixed-lineage leukemia protein-5 (MLL5), proliferating cell nuclear antigen, heparan sulfate proteoglycans, nidogen-1, and platelet-derived growth factor-DD [16] . Recently, we developed a novel CAR that targets NKp44 ligands and reported that T cells transduced with this CAR showed antitumor effects against various pediatric solid tumors, including some sarcomas [17] . Ligands for NKp44 are only expressed in malignant tumor cells, and not in normal tissues [16] .…”

NK Cells Armed with Chimeric Antigen Receptors (CAR): Roadblocks to Successful Development

“…It has been shown that hinge and transmembrane domains can alter the functionality of a CAR by regulating the threshold and amount of CAR signaling, respectively [ 51 , 52 ]. Moreover, they affect the expression levels of the CAR and thereby the antigen recognition [ 53 ].…”

T Cell Activation Machinery: Form and Function in Natural and Engineered Immune Receptors

“…These were effective in mouse models of multiple myeloma and ovarian cancer expressing stress ligands recognized by NKG2D [ 162 , 163 , 164 ] but a phase I clinical trial showed limited NKG2Dζ CAR-T cell expansion and persistence [ 165 ], indicating that improvements are likely to be required for clinical viability. Others have investigated similar fusions using NKp44 and NKp30 [ 166 ], activating receptors that associate with DAP12 or ζ through their TM domains, respectively [ 25 ], to endow T cells with NK-like specificity. A related approach has been used to generate scFv-based CARs with the spacer and TM domains from activating NK cell receptor (killer cell immunoglobulin like receptor, two Ig domains and short cytoplasmic tail 2, KIR2DS2 )—KIR-CARs [ 167 ], which assemble with DAP12 through their TM domains [ 61 , 62 , 168 ] ( Figure 3 ).…”