BACKGROUND The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. METHODS To further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. RESULTS In Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions. CONCLUSION This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1-expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.