Abstract Background: In the clear cell renal cell carcinoma (ccRCC) tumor microenvironment (TME), transforming growth factor-β (TGF-β) is a dominant immunosuppressive cytokine that suppresses NK metabolism, proliferation, and cytotoxicity. Rather than blocking TGFβ receptor, we hypothesized that elevated TGFβ in the ccRCC could be harnessed as a trigger to drive local, therapeutic cytokine expression. Methods: We engineered TGFβ-sensing circuits to conditionally express IL-12A3—a collagen-binding IL-12 fusion cytokine, in response to tumor derived TGFβ. The initial forward-strand designs produced TGFβ-inducible 70CAR/IL-12, but with leaky IL-12 expression. However, a bidirectional layout (sensor+IL-12A3 on the reverse, 70CAR on the forward) cut baseline IL-12 expression by ∼87% (43 vs 362 pg/mL, p=0. 0019) without affecting CAR expression or NK cell function. To boost efficacy, we added constitutive IL-18 downstream of 70CAR (70CAR-12A3/18), while IL-12 remained TGFβ-regulated. Primary NK cells were transduced and assessed in vitro for activation and cytotoxicity against A498, ACHN, and CD70+ ccRCC PDXs, and in vivo in NSG-IL15 mice bearing A498 tumors. Tumor growth, NK persistence/infiltration/activation, and safety were evaluated, including body weight and day 7 serum cytokines (IL-12, IL-18, IL-1β, IL-2, IL-6, IFNγ, GM-CSF). Results: High transduction efficiency was achieved in NK cells after transduction with constructs 70CAR (45 ∼ 78%, n=8), 70CAR-12A3 (36 ∼ 62%, n=8), 70CAR-18 (41 ∼ 80%, n=8), and 70CAR-12A3/18 (27 ∼ 55% n=8) at MOI=2. 70CAR-12A3/18 NK cells secreted high IL-18 (∼1, 800 pg/mL) and TGFβ-inducible IL-12 (21. 6 vs 324. 2 pg/mL ± TGFβ1; p=0. 0013), showed enhanced cytotoxicity against A498, ACHN, and CD70+ ccRCC PDXs, and fully overcame TGFβ-mediated suppression with combined IL-12+IL-18 (either alone was insufficient). In NSG-IL15 mice subcutaneously implanted with A498 tumors, 70CAR-12A3/18 significantly reduced tumor volume (p0. 0001) and extended survival (p0. 0001). It yielded the highest human NK cell percentages among groups in blood at day 14 and, at endpoint, across blood, bone marrow, liver, lung, spleen, and tumor of treated mice. In serum, human IL-12 reached up to 34 pg/mL in 70CAR-12A3/18 or 70CAR-12A3 treated mice, while IL-18 averaged ∼1, 300 pg/mL (70CAR-12A3/18) and ∼2, 300 pg/mL (70CAR-18). No CRS-related cytokines were significantly elevated. Conclusion: We present a bidirectional TGFβ-sensing circuit that pairs constitutive IL-18 with TGFβ-inducible IL-12 for context-specific cytokine delivery and enhanced CAR NK activity. 70CAR-12A3/18 NK cells reduced tumor burden and extended survival in NSG mice bearing ccRCC xenografts without causing systemic toxicity. This modular TGFβ-sensor is generalizable, offering a blueprint for NK therapies targeting immunosuppressive TMEs in solid and hematologic tumors. Citation Format: Fuguo Liu, Xingyu Deng, Veronica W. Hui, Shikha Gupta, Wenxin Xu, Maily Nguyen, Mubin Tarannum, Andreia Maia, Shaobo Yang, Stephanie Sendker, Alaa K. Ali, John Koreth, Jose A. Cancellas, Jianzhu Chen, Robert Soiffer, Catherine Wu, Jerome Ritz, Toni K. Choueiri, Rizwan Romee. Engineering novel CAR NK cells to overcome TGF-β suppression in renal cell carcinoma abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₂): Abstract nr A019.
Liu et al. (Fri,) studied this question.