In vitro modelling of local gene therapy with IL-15/IL-15R and a PD-L1 antagonist in melanoma reveals an interplay between NK cells and CD4⁺ T cells

Blockade of the immune checkpoint axis consisting of programmed death-1 (PD-1) and its ligand PD-L1 alleviates the functional inhibition of tumor-infiltrating lymphoid cells yet weakly induces their expansion. Exogenous cytokines could further expand lymphoid cells and thus synergize with alpha PD-L1 therapy. However, systemic delivery of most cytokines causes severe toxicity due to unspecific expansion of immune cells in the periphery. Here, we modelled local delivery of cytokines and alpha PD-L1 therapeutics to immune cell-containing in vitro melanoma tumors. Three-dimensional tumor models consisting of 624-MEL cells were co-cultured with human peripheral blood lymphoid cells (PBLs) in presence of the cytokines IL-2, IL-7, IL-15, IL-21 and IFN-gamma. To model local gene therapy, melanoma tumors were modified with lentiviral vectors encoding IL-15 fused to IL-15R alpha (IL-15/IL-15R alpha) and K2-Fc, a fusion of a human PD-L1 specific single domain antibody to immunoglobulin (Ig)G1 Fc. To evaluate the interplay between PBL fractions, NK cells, CD4(+) T cells or CD8(+) T cells were depleted. Tumor cell killing was followed up using real time imaging and immune cell expansion and activation was evaluated with flow cytometry. Among the tested cytokines, IL-15 was the most potent cytokine in stimulating tumor cell killing and expanding both natural killer (NK) cells and CD8(+) T cells. Gene-based delivery of IL-15/IL-15R alpha to tumor cells, shows expansion of NK cells, activation of NK cells, CD4(+) and CD8(+) T cells, and killing of tumor spheroids. Both NK cells and CD8(+) T cells are necessary for tumor cell killing and CD4(+) T-cell activation was reduced without NK cells. Co-delivery of K2-Fc improved tumor cell killing coinciding with increased activation of NK cells, which was independent of bystander T cells. CD4(+) or CD8(+) T cells were not affected by the co-delivery of K2-Fc even though NK-cell activation impacted CD4(+) T-cell activation. This study demonstrates that gene-based delivery of IL-15/IL-15R alpha to tumor cells effectively mediates anti-tumor activity and sensitizes the tumor microenvironment for therapy with alpha PD-L1 therapeutics mainly by impacting NK cells. These findings warrant further investigation of gene-based IL-15 and K2-Fc delivery in vivo.