Cell therapy could benefit from BCG: how anti-tumour BCG-primed NK cells selectively proliferate over other lymphocytes and efficiently kill cancer cells

Abstract Natural killer (NK) cell-based immunotherapies are safe, promising treatments for patients with cancer. Nevertheless, the short-lived nature of NK cells, the heterogeneity of NK populations and the need to infuse large number of cells for efficient tumour elimination represent important challenges for the development of NK cell-based therapies. Therefore, improved approaches to produce high numbers of long-lived, cytotoxic NK cells are essential for clinical applications. We have previously identified anti-tumour NK cells (CD56highCD16+NKG2A+) generated upon exposure to Bacillus Calmette-Guérin (BCG), an early immunotherapy that has been used for decades to treat bladder cancer. Here, we describe that BCG-primed NK cells (B-pNK) kill a broad range of solid tumours and specifically proliferate, without the need of cell sorting, by weekly stimulations with low doses of cytokines, while BCG and other peripheral blood mononuclear cells decrease in the culture. In depth scRNA-seq analysis showed that B-pNK upregulated IL12, IL15 or IL21 signalling pathways, but not IL18, according to the Reactome database. Consequently, B-pNK cells potently proliferated and enriched over other lymphocytes, after weekly stimulations with this cytokine combination, used in low concentration to avoid overstimulation. Despite NKG2A expression, B-pNK mediated potent anti-tumour activity as demonstrated in functional assays, including degranulation, specific cytotoxicity and intracellular-cytokine release, being NKG2D the main activating receptor involved in tumour recognition and elimination. We propose that a strategy based on BCG-priming of NK cells provides a scalable and economical approach for the development of safe and universal cellular immunotherapies against solid tumours.