Abstract A089: Improving Therapeutic Window of Claudin 18.2-Targeted CAR-T cells in Pancreatic Cancer

Abstract Claudin 18.2 (CLDN18) targeted therapies, such the CAR-T cell, Satri-cel (CT041) are expanding access to immunotherapy in pancreatic cancer, however, high rates of nausea and vomiting were seen thought to be related to on target off tumor (OT/OT) toxicity in the stomach, a known site of normal CLDN18.2 expression. Furthermore, their utility is limited by relatively short duration of response. Taking advantage of the high degree of homology of CLDN18.2 between mouse and human we develop novel, fully-human, nanobody-based CARs with superior therapeutic window compared to CT041, extending efficacy and reducing OT/OT. We also demonstrate toxicity inversely correlates with CAR binder affinity. Following an antibody discovery campaign, novel fully-human heavy chain only binders (HCAb) specific to CLDN18.2; cross reactive to both the human and mouse protein, were cloned into the 4-1BB containing CAR. In vitro screening identified CARs with two highly active binders, “5795-VH” and “5797-VH”. The binding kinetics of IgG reformatted versions of binders 5795, 5797 and CT041 were determined via SPR against both mouse and human CLDN18.2. CT041-IgG had highest affinity to human CLDN18.2 (KD=3.64±0.2 nM), 5797-IgG had slightly lower affinity (KD=4.5±3nM) while 5795-IgG had ∼10-fold lower affinity (KD=21.8±2nM). Using the human pancreatic cancer xenograft model of PATU8988s, cells were allowed to engraft and expand in NSG DKO mice, then treated with a single dose of either 3 x105 or 1 x106 5795-VH, 5797-VH, or CT041-scFv CAR-T and compared to 1 x106 BCMA-scFv irrelevantly targeted control treated animals. All animals treated with either CT041, or 5797 based CAR-T succumbed to toxicity, even at the lower dose, while 5795 based CAR-T had long term tumor control at both doses, with minimal toxicity in this tumor model (mOS 31-36d for CT041-scFv groups and mOS not reached by 60d for 5795-VH groups; p<0.01). Stomach was harvested from animals in this model and stained by multiplex immunofluorescence and H&E. Animals treated with CT041-scFv CAR showed the highest degree of CAR-T cell infiltration into the stomach, followed by 5797-VH. then 5795-VH CAR-T. This also correlated with degree of tissue disruption, with almost complete atrophy and erosion in the CT041-scFv treated group, only around 1% atrophy and some erosion in the 5797-VH treated animal, very little if any tissue disruption in the 5795-VH animal and normal stomach architecture in the BCMA-scFv control. T cell infiltration into the stomach was also evident regardless of tumor bearing status in animals treated with CT041-scFv. A primary challenge to extending immunotherapies to solid tumors is selecting antigens with widespread tumor expression, but limited expression in normal tissues. We demonstrate that appropriate modeling of anticipated toxicity and careful examination of binding kinetics is critical to engineering a CAR with an optimal therapeutic window and share a novel design of CAR-T for targeting CLND18.2 in pancreatic cancer. Citation Format: Elizabeth J. Carstens, Kazuki Takahashi, Martina De Vizio, Micaela Morgado, Shahryar Khoshtinat Nikkhoi, Abhishek Mangipudi, Canh Nguyen, Tate Weltzin, Qiang Lv, Jue Zeng, Cui Nie, Chiangjing Deng, Xiaoxiao Wang, Lile Liu, Samuel J. Klempner, Anusuya Ramasubramanian, Jonathan A. Nowak, Andrew Aguirre, Eric L. Smith. Improving Therapeutic Window of Claudin 18.2-Targeted CAR-T cells in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A089.