Abstract Introduction: Claudin 18.2 (CLDN18.2) is highly expressed in gastric cancer with minimal expression in normal tissues, making it an attractive target for engineered cell therapies. CAR NK cells offer a potentially lower risk of inciting inflammatory cytokines and may be easily sourced for allogeneic or “off-the-shelf” use. However, conventional CAR NK approaches often show limited persistence and reduced activity when engaging with solid tumors. To enhance antitumor activity and persistence, we developed CLDN18.2 nanobody-based CAR NK cells incorporating IL-15/IL15sushi armoring. This study evaluated the antitumor efficacy and persistence in vitro and in vivo of the armored CLDN18.2 CAR NK cell construct generated via a feeder-independent NK expansion platform. Methods: Alpacas were immunized with recombinant human CLDN18.2 extracellular domain protein. Peripheral blood mononuclear cell (PBMC) mRNA was extracted and converted into a VHH (nanobody) to generate a library. The library was panned against CLDN18.2 antigen to enrich for high-affinity binders, which were identified through ELISA screening and cellular binding analysis by flow cytometry. VHH clone sequencing were used to generate our cCAR.A CLDN18.2-specific nanobody was identified through screening and used to generate an armored CLDN18.2 CAR NK cell construct. NK cells were isolated from cryopreserved cord blood and expanded using a feeder-independent NK expansion platform. CAR expression was measured by flow cytometry. Cytotoxicity was assessed using SNU-601 and REH-C18.2xp target cells. NSG mice received CAR NK cells. Results: The armored CLDN18.2 CAR NK cells displayed strong CAR expression following NK cell transduction. CLDN18.2 CAR NK cells demonstrated remarkable in vitro cytotoxicity across multiple effector-to-target ratios, eliminating CLDN18.2-positive targets rapidly and completely. In vivo, the armored CLDN18.2 CAR NK cells were observed to impart strong anti-tumor activity resulting in regression in SNU-601 xenografts. In NSG mice, armored CLDN18.2 CAR NK cells resulted in rapid tumor reduction and measurable NK persistence in mouse peripheral blood. Conclusions: Armored CLDN18.2 nanobody CAR NK cells generated via a feeder-independent NK expansion platform showed strong antitumor activity and improved in-vivo performance in CLDN18.2-positive tumor models. CAR NK cells also demonstrated sustained persistence following infusion. Together, these findings support future clinical investigation of our platform in patients with CLDN18.2-positive gastric cancer. Citation Format: Byeong-Hyeok Choi, Ga-Ram Hwang, Morgan Flaherty, Vincent M. DeStefano, Masayuki Wada, Kevin Pinz, Jennifer E. Chow, Nabil Hagag, Yu Ma, Jing Luo, Yupo Ma. Preclinical study of armored claudin 18.2 nanobody CAR NK cells targeting gastric cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6717.
Choi et al. (Fri,) studied this question.