The gamma delta T/NK cell product GADEKILL as a novel immunotherapeutic tool for neuroblastoma patients: role of B7H6 and BTN2A1 in tumor cell killing.

BACKGROUND: Anti-GD2 monoclonal antibody effectively treats high-risk neuroblastoma (HR-NB) by recruiting NK cells for antibody-dependent cellular cytotoxicity (ADCC). We recently developed a cell product containing mature, cytotoxic γδ T and NK cells (GADEKILL), and its potential use as a novel immunotherapy for HR-NB has been investigated. METHODS: The GADEKILL γδ T and NK cells were analyzed by flow cytometry for the expression of activating and inhibitory receptors and for cytotoxicity against NB, both with and without dinutuximab-β, at a 1:1 effector-to-target ratio. NB cell lines with high and low/absent GD2 expression, as well as patient-derived 3D tumor spheres, all GD2-expressing, were used as targets. Comparative analyses were performed between GADEKILL NK and purified NK cells obtained from the same donor leukapheresis. Furthermore, a panel of NB cell lines was tested for the expression of B7H6 (i.e., NKp30 ligand), Human influenza hemagglutinin-tag (HA-TAG) and calreticulin (i.e., NKp46 ligands), and butyrophilin (BTN)2A1 and BTN3A1/2/3 (i.e., TCRVδ2 ligands), and the impact on GADEKILL cytotoxicity was assessed. RESULTS: Compared to their purified counterparts, GADEKILL NK cells showed: (i) higher expression of NKp30 and NKp44 and lower expression of CD16 and NKG2D, (ii) greater cytotoxicity (CD107a(+)) against GD2(-) NB cells, (iii) stronger induction of lysis in low GD2-expressing NB cells and patient-derived 3D tumor spheres, and (iv) comparable ADCC. In addition, both γδ T and NK cells degranulated and consistently induced lysis in a panel of NB cell lines and patient-derived 3D tumor spheres expressing B7H6, calreticulin, HA-TAG, BTN2A1, and BTN3A1/2/3 consistently. Finally, NB cell lysis positively correlated with B7H6 and BTN2A1, and B7H6-blocking experiments revealed a significant decrease in target cell lysis when cells highly expressing B7H6 were used as targets. CONCLUSIONS: Our study demonstrated the potential antineuroblastoma activity of the GADEKILL, supporting its therapeutic use, particularly in the context of relapsed/refractory R/R HR-NB with low GD2 expression.