Abstract A019: Engineering novel CAR NK cells to overcome TGF-β suppression in renal cell carcinoma

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 (p