Abstract 870: Engineering of a novel Buddy-CAR system: CAR-NK cells expressing IL7/IL7Rα exhibit superior proliferation and anti-tumor responses while promoting trans-activation of the bystander T cells

Abstract Natural killer (NK) and chimeric antigen receptor (CAR) NK cells offer a promising platform for cellular immunotherapy. However, the immunosuppressive tumor microenvironment (TME), particularly in pancreatic cancer, negatively affects the efficacy of cell therapies. IL7 is an attractive cytokine to module TME as it promotes CD4+ and CD8+ T cell proliferation while maintaining them in a less differentiated state, improving metabolic profile, and preventing exhaustion. Mature NK cells are minimally responsive to IL7 as they lack the expression of IL7Rα. To overcome this, we engineered the Buddy-CAR system, consisting of CAR-NK cells expressing IL7Rα AND secreting IL7 to enhance the activation and proliferation of CAR-NK cells (cis-activation) and modulate the TME by activating the bystander tumor-infiltrating T cells. We designed three CAR constructs: (1) Mesothelin (MSLN)-CAR (M), (2) MSLN-CAR secreting IL7 (M7) and (3) MSLN-CAR expressing IL7Rα AND secreting IL7 (M7R). Peripheral blood-derived NK cells were transduced using Baboon lentivirus achieving a high transduction efficiency and IL7 secretion was assessed by ELISA. M7R cells exhibited a superior proliferation index of 1.9 versus 1.1 for M (p=0.03) on day 9 using CTV flow cytometry assay. M7R showed increased cytotoxicity against MSLN+ ASPC1 cells, 61.4% vs 52.3% for M7 (p=0.712) and 49.8% for M7R (p=0.002) at a 2:1 ratio. In addition, M7R showed a 3.9-fold increased IFN-γ production versus M7 (0.9, p=0.0002) and M (1, p=0.0005). The incorporation of IL7Rα and IL7 also led to increased expression of NKp30, NKp46, pSTAT1 and pSTAT5. We then evaluated the Buddy-CAR system’s ability to stimulate bystander T cells by co-culturing IL7-releasing CAR-NK cells (M7 and M7R) with donor-matched MSLN-CAR T cells for 9 days in a 1:1 ratio. In a CTV flow cytometry assay, CAR-T cells showed a higher percentage of cells in >G3 when co-cultured with M7 (26.3%, p=0.048) and M7R (27.6%, p=0.035), versus M (2.72%). Lastly, we assessed the impact of IL7 signaling in CAR NK cells in vivo by injecting 6x106 of M7 and M7R cells into NGS-Tg (Hu-L15) mice intravenously. Mice injected with M7R showed a significantly higher percentage of human CD45+ infiltrating cells in the bone marrow (47.6%, p=0.02), liver (66.7%, p<0.0001), and spleen (69.8%, p=0.03), two weeks post-injection compared to M7-NK cells (<3%), indicating superior trafficking and proliferation in vivo. Ongoing studies are evaluating tumor control and effects on T cell proliferation and trafficking of the Buddy-CAR system in a xenograft mouse model. The versatility of the Buddy-CAR system allows for targeting different antigens by modifying CAR specificity while also modulating the immunosuppressive TME, offering a powerful approach to significantly improve the efficacy of CAR-based therapies across diverse cancer types. Citation Format: Andreia Maia, Mubin Tarannum, Sara Piccinelli, Mila Stanojevic, Maily Nhuyen, Grace Birch, Alaa Ali, Rizwan Romee. Engineering of a novel Buddy-CAR system: CAR-NK cells expressing IL7/IL7Rα exhibit superior proliferation and anti-tumor responses while promoting trans-activation of the bystander T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 870.