The effectiveness of immunotherapeutic mAbs or CAR-T therapies can be hindered by tumor cells losing antigen expression and upregulating neoantigens and immune checkpoint (IC) ligands. In contrast, NK cells are not dependent on specific antigens for cytotoxicity, offering promise in cellular immunotherapy. Allogeneic NK cells have a lower risk of graft-versus-host disease (GvHD) and cytokine release syndrome (CRS), making them a safer, "off-the-shelf" option. However, a hostile tumor microenvironment can lead to resistance and reduce NK cell efficacy. To enhance their therapeutic potential, NK cells can be genetically engineered to target tumor markers common to a broad range of tumors (as IC ligands) and to overcome the hostile TME.

NK cells, isolated from peripheral blood of healthy donors, were genetically modified using retroviral vectors. The retroviral tri-cistronic vectors encode chimeric proteins composed of IC extracellular domains (specifically extracellular PD1- exPD1) fused to the cytoplasmic NKG2D domain linked to 4.1BB co-stimulatory in frame with NKG2A single chain variable fragment (scfv) and IL15.

exPD1-NK cells, obtained upon transfection, increased the cytotoxicity against PD-L1+ tumor cells. Moreover, exPD1-NK cells constitutively release NKG2A-scfv fragment and IL15. The NKG2A-scfv fragments disrupted the NKG2A/HLA-E interaction and blocked the NK inhibitory receptor NKG2A signal in the TME. In addition, the release of IL15 supported the NK cell survival and proliferation in vivo. The ability to release NKG2A scfv and IL15 makes exPD1-NK cells better able to kill even PD-L1 negative cells.

Our findings provide a comprehensive strategy to overcome significant obstacles in NK cell therapy, demonstrating the potential of these innovations to improve the effectiveness of NK cell-based immunotherapies and offer new therapeutic options for patients with solid tumors. This research could contribute to a paradigm shift in cancer treatment.