45Tumor matrix directed IL12/A3 fusion cytokine secreted by CD70-CAR NK cells markedly enhances anti-ccRCC responses with limited systemic exposure

Abstract Background Despite recent advances, metastatic clear cell renal cell carcinoma (ccRCC) remains largely incurable in most patients, and novel immunotherapeutic strategies to address checkpoint inhibitor refractory renal cell carcinoma are needed. Natural killer (NK) cells are immune effector lymphocytes that are specialized in the cytotoxic elimination of cancer cells. Chimeric antigen receptor (CAR) NK cells have also shown promising effects in clinical trials. IL-12 is a potent cytokine that mediates type 1 immunity and antitumor responses but with systemic toxicity. We hypothesize that IL-12 secreted by CD70-CAR NK cells will allow “tumor-directed” IL-12 delivery and with the incorporation of a novel motif, collagen-binding domain A3, we will further minimize toxicity. Methods We engineered lentivectors to express CD70-CAR, CD70-CAR+IL-12, and CD70-CAR+IL-12/A3, and evaluated their transduction efficiencies in human NK cells. The phenotypes of IL-12-secreting CD70-CAR NK cells were analyzed using flow cytometry and bulk RNA sequencing. Untransduced (UTD) and modified NK cells were cocultured with ccRCC tumor cell lines (A498 and ACHN) at various effector to target (E: T) ratios to assess target cell lysis. NK cell activation was determined by measuring degranulation (CD107a) and IFNγ expression. IFNγ secretion was quantified from coculture supernatants. The killing of ccRCC PDXs was similarly evaluated. In vivo, the therapeutic efficacy of CD70-CAR+IL-12/A3 NK cells was tested using ccRCC xenograft models using A498 cells injected subcutaneously into NSG mice. The mice were divided into five groups: no treatment (PBS), NK cell, CD70-CAR NK cell, CD70-CAR+IL-12 NK cell, and CD70-CAR+IL-12/A3 NK cell. When tumors reached ∼100 mm³, 1x10^6 NK cells were administered intravenously. Tumor volumes and mouse survival were monitored. For safety evaluation, the animals were closely monitored for their weight and wellbeing and plasma collected on day 7 post CAR NK cell injection to evaluate the production of IL-12 and other key inflammatory cytokines including IL-1β, IL-2, IL-6, IFNγ, IL-18 and GM-CSF that are associated with cytokine release syndrome (CRS). Results High transduction efficiency was achieved in the NK cells with all three CAR constructs (50–85%). IL-12-engineered CD70-CAR NK cells exhibited significantly increased surface expression of CD25, LFA1, CXCR4, and sLex compared to regular CD70-CAR NK cells. Gene expression analysis revealed upregulation of IL12A, IL12B, IFNG, GZMB, IRF1, SOCS3, and ABCA1, alongside downregulation of CISH. These cells also displayed enhanced metabolic fitness, with an increased oxygen consumption rate (OCAR). CD70-CAR NK cells that secrete IL-12 and IL-12/A3 exhibited significantly increased cytotoxicity in vitro against ccRCC tumor cell lines A498 and ACHN, as well as CD70+ ccRCC patient-derived xenografts (PDXs). The presence of IL-12 and IL-12/A3 effectively stimulated NK cell degranulation and IFNγ production following coculture with ccRCC tumor cells. In vivo studies showed that CD70-CAR NK cells secreting IL-12 and IL-12/A3 achieved notably better control of ccRCC tumors compared to CD70-CAR NK cells alone. Mice treated with CD70-CAR+IL-12/A3 NK cells demonstrated better survival rates than those treated with CD70-CAR+IL-12 NK cells or other constructs (Fig. 1). Additionally, CD70-CAR+IL-12/A3 NK cells resulted in significantly lower systemic levels of IL-12 7 days after CAR NK cell infusion compared to CD70-CAR+IL-12 NK cells, suggesting that fusing IL-12 to a collagen binding domain can reduce systemic IL-12 exposure. Conclusions Incorporating IL-12/A3 into CAR NK cells is feasible, safe, and significantly enhances their cytotoxicity against ccRCC tumor cells in vitro and in vivo. These findings provide strong rationale for further clinical evaluation of CD70-CAR+IL-12/A3 NK cells in patients with advanced ccRCC.