Chimeric antigen receptor (CAR)-T and CAR-NK cell therapies have shown promise in cancer treatment, but their effectiveness remains hampered by the immunosuppressive tumor microenvironment (TME). IL-7 is a promising cytokine for TME modulation, as it promotes T cell proliferation and prevents exhaustion. However, mature Natural Killer (NK) cells are unresponsive to IL-7 due to the absence of IL-7Rα. To address this limitation, we engineered CAR-NK cells that co-express IL-7Rα and secrete IL-7, aiming to achieve autocrine activation of CAR-NK cells and paracrine stimulation of tumor-infiltrating T cells within the TME.

We developed three CAR-NK cells: CAR-NK cells (NK-0), CAR-NK cells secreting IL-7 (NK-7), and CAR-NK cells secreting IL-7 and expressing IL-7Rα (NK-7R). To evaluate the versatility of our approach, two CAR-NK cells were designed to target either CD70 or Mesothelin (MSLN), two clinically relevant tumor antigens. NK-7R exhibited increased cytotoxicity against MSLN+ tumor cells (61.4% vs 52.3% for NK-7, p=0.712 and 49.8% for NK-0, p=0.002) at a 2:1 ratio. NK-7R produced higher levels of IFN-γ, with a 3.9-fold increase compared to NK-7 (0.9, p=0.0002) and NK-0 (1, p=0.0005). In proliferation assays using CellTrace Violet (CTV), NK-7R demonstrated superior proliferative capacity, with an improved proliferation index (1.9 vs 1.1 for NK-0, p=0.03). Metabolic profiling revealed that NK-7R cells exhibited enhanced metabolic fitness, with superior glycolytic activity (17.57 mpH/min vs M=4.06 mpH/min, p=0.01 and M7=2.53 mpH/min, p=0.006) and superior maximum respiration (524 pmol/min vs M=171.02 pmol/min, p=0.03 and M7=196.86 pmol/min), indicating superior glycolysis and mitochondrial respiration capacity.

To assess the impact on T cell function, CAR-T cells were co-cultured with the different CAR-NK cells. In these experiments, CD19-T cells were developed to evaluate the specific killing and proliferation of CAR-T cells. CD19-T cells demonstrated higher proliferation in the presence of IL-7-secreting CAR-NK cells, with 47.5% of CTV-neg T cells when co-cultured with NK-7 (p=0.023) and 44.1% with NK-7R (p=0.032) compared to 2.37% with NK-0, at a 1:1 ratio. Furthermore, CD19-T cells showed increased cytotoxicity against lymphoma (Raji) cells when co-cultured with IL-7-secreting CAR-NK cells (0.53 normalized to target vs 0.31 for CD19-T (p=0.0346), 0.36 for CD19-T plus NK-0 and 0.56 for CD19-T plus NK-7).

In vivo, the trafficking and persistence of CAR-NK cells were evaluated in NGS-TG (Hu-IL15) mice (n=5) following intravenous (IV) injection. Two weeks following injection, NK-7R cells demonstrated higher infiltration of hCD45+ CAR-NK cells in multiple organs compared to NK-0, including bone marrow (0.23% vs 0.05%, p=0.0039), liver (2.17% vs 0.04%, p=0.0477), spleen (1.40% vs 0.05%, p=0.0367), lungs (5.19% vs 0.31%, p=0.0021), and blood (1.38% vs 0.27%, p=0.0280), indicating superior trafficking and proliferation in vivo. Furthermore, in NGS-TG (Hu-IL15) mice (n=5), co-administration of CD19-T cells with NK-7R resulted in superior tumor control of Raji cells on day 21, as measured by area under the curve (AUC) of total flux [p/s]: 2.14 x 108 for CD19-T plus NK-7R, compared to 2.15 x 109 for CD19-T plus NK-7, 4.41 x 109 for CD19-T plus NK-0, and 2.36 x 109 for CD19-T cells alone. Ongoing in vivo experiments are evaluating the impact of NK-7R on overall survival in this model.

Mechanistically, NK-7R cells exhibited significantly superior phosphorylated STAT1 (pSTAT1) levels (5.14 vs 1 for NK-0, p=0.00468 and 0.85 for NK-7, p=0.0389) by western blot analysis. Bulk RNA-sequencing revealed upregulation of STAT1, IFNG, CXCL10, IFIT1, IFIT3, and TNFSF10 in NK-7R compared to NK-7 and NK-0. These findings indicate that, in contrast to T cells, where IL-7/IL-7Rα signaling primarily activates the STAT5 pathway, the IL-7/IL-7Rα axis in NK cells predominantly signals through the STAT5 but also STAT1/CXCL10/IFNG pathways.

In summary, our data demonstrate that engineering CAR-NK cells to co-express IL-7Rα and secrete IL-7 enhances their cytotoxicity, proliferation, metabolic fitness, and in vivo persistence, while also supporting T cell function and remodeling the TME. This dual autocrine and paracrine strategy represents a novel and promising approach to overcoming immunosuppressive tumors and improving the efficacy of adoptive cell therapies across multiple cancer types.

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2025
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