Dataset Information

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.

ABSTRACT:

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.

SUBMITTER: Morandi F

PROVIDER: S-EPMC12901508 | biostudies-literature | 2026

REPOSITORIES: biostudies-literature

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Publications

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.

Morandi Fabio F Della Lastra Martina M Pastorino Fabio F Ciampi Eleonora E Faraci Maura M Brignole Chiara C Giardino Stefano S Airoldi Irma I

Frontiers in immunology 20260130

<h4>Background</h4>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.<h4>Methods</h4>The GADEKILL γδ T and NK cells were analyzed by flow cytometry for the expression of activating and inhibitory receptors and for cytot ...[more]

PMID: 41694338

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Project description:Gamma-delta T cells are potent anti-cancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or HLA background. Gamma-delta T cells can sense conserved cell stress signals prevalent in transformed cells, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vg9Vd2 T cells – the most abundant subset of human gamma-delta T cells – recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1, a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here, we layered genome-wide CRISPR screens in target cancer cells to identify pathways that regulate: (1) killing by gamma-delta T cells during co-culture, and (2) BTN3A cell surface expression. In addition to confirming the importance of the phosphoantigen-producing mevalonate pathway, the screens revealed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of gamma-delta T cells through transcription, post-translational modifications, membrane trafficking. Notably, IRF1 and ZNF217 were discovered to be positive and negative transcriptional regulators that directly occupy the promoters of BTN3A1/2/3 genes. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of BTN3A, as well as BTN2A1, during metabolic crises was dependent on AMP-activated protein kinase (AMPK). Finally, small molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vg9Vd2 TCR-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of gamma-delta T cell stress surveillance and suggests new avenues to enhance gamma-delta T cell anti-cancer activity.

Dataset Information

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

Methods

Results

Conclusions

Publications

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.

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