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.