Long-term cytotoxic NK cells with broad anti-tumour capacity proliferate selectively, without exhaustion, after BCG priming and extremely low doses of cytokines

Background: Natural killer (NK) cell-based immunotherapies, currently under investigation, appear to be safe, efficient treatments in patients with haematological tumours. Nevertheless, the short-lived nature of these cells combined with the need to infuse large number of cells for efficient tumour elimination represent important challenges for the development of NK cell-based therapies. Although NK cell anti-tumour activity is regulated by cytokines, constant stimulation together with the immunosuppressive tumour environment can result in NK cell exhaustion. Therefore, improved approaches to produce highly cytotoxic and longer-lived NK cells are of considerable clinical interest. Methods: Peripheral blood mononuclear cells (PBMC) are primed in vitro with a pulse of either Bacillus Calmette-Guérin (BCG) vaccine or a cell wall extract of M. bovis , followed by weekly stimulations with low doses of IL12, 15 and 21. The phenotype and anti-tumour fitness of the activated NK cell culture were examined using scRNA-seq, flow cytometry and functional assays, including degranulation, specific cytotoxicity and IFNγ release. Results: we describe a novel strategy for the generation of long-lived activated NK cells capable of killing a broad range of solid tumours. A unique subset of cytotoxic NK cells (CD56high CD16+ NKG2A+) specifically proliferated in vitro , and was further expanded without functional exhaustion under minimal survival cytokine combinations. Mycobacterial cell-wall fractions also activated NK cells that recognised tumours efficiently, and proliferated well, and this approach has the advantage that no live bacteria are present in the cultures. Conclusions: We propose that BCG-priming to expand anti-tumour NK cells, without cell sorting, could be a scalable and economical basis for the development of safe and universal cellular immunotherapies against solid tumours. Key messages Adoptive therapy with sorted NK cells grown in IL12, 15, 18 are being tested in clinical trials, but are only efficient for haematological tumours. In addition, their survival in vivo is limited. Here, we define culture conditions that drive the selective proliferation of long-lived natural killer (NK) cells, without the need of cell sorting, in minimal doses of cytokines, after priming with BCG or mycobacteria components. BCG-primed NK cells grow and maintain effective cytotoxic function against a variety of solid tumours in vitro , without exhaustion for at least 28 days of culture. This new approach provides the basis for the generation of innate adoptive cell therapy tools. ### Competing Interest Statement The authors have declared no competing interest.