Abstract 5914: Development of immunohistochemistry detection kits for claudin family: CLDN6, CLDN9, and CLDN18.2
Authors: Qingbo Wang, Xinyue Wang, Jing Tao, Tongyu Xiao, Xiao Wang, Minyang Li, Xiao Lv, Wenwen Zhao, Jiayue Geng, Yixin Liu, Zhanjiao Yu, Yuanhao Li
Journal: Cancer Research (2025)

Background: Claudins are a family of integral membrane proteins that are critical for the formation of tight junctions in epithelial and endothelial cells. Aberrant expression of Claudin family members is implicated in the progression of various cancers, making them valuable biomarkers for tumor diagnosis, prognosis, and therapeutic targeting. such as CLDN-6, CLDN-9, and the splice variant CLDN-18.2. An antibody drug targeting CLDN18.2 was recently approved by the FDA for the treatment of advanced gastric cancer. Despite their clinical potential, routine assessment of these proteins in cancer diagnostics is limited because of the lack of highly specific detection tools. we developed novel immunohistochemistry (IHC) detection kits for CLDN-6, CLDN-9, and CLDN-18.2 to facilitate their use as diagnostic and therapeutic biomarkers. Methods: We used AI-designed peptides from the CLDN-6, CLDN-9, and CLDN-18.2 proteins conjugated with keyhole limpet hemocyanin (KLH) as immunogens to immunize the BABL/c mice. The initial IHC antibody candidates were identified by ELISA and further optimized using various structurally facilitated AI approaches. The selected antibodies were evaluated by IHC in formalin-fixed paraffin-embedded (FFPE) CHOK1 cells expressing full-length CLDN6, 9, 18.2 or CLDN 3, 4, 18.1. These IHC antibodies were also subjected to ELISA, Western blotting, and tumor tissue microarray IHC staining to determine the features of IVD products. Furthermore, we conducted intra-day, inter-day, and inter-operator studies to assess the consistency of IHC staining under the conditions of our established system. Results: The final IHC antibody clones obtained for CLDN6, CLDN9, and CLDN18.2 were #7, #10, and #9B3, respectively. ELISA tests showed that they all had good binding affinity. Tumor tissue microarray staining showed that these antibodies can specifically stain the target protein in paraffin samples from testicular, endometrial, gastric cancer, and other tumor tissues. The WB assay and IHC staining results confirmed their binding specificity. We established a preliminary reaction system for the IHC assay with final working concentrations of 0.25 μg/ml, 0.25 μg/ml, and 20 μg/ml for #7, #10, and #9B3, respectively. The three IHC assays showed good intra-day, inter-day, and inter-operator reproducibility under the current reaction system, demonstrating good robustness as IHC diagnostic products. Conclusions: We successfully developed IHC antibodies against CLDN6, CLDN9, and CLDN18.2. These antibodies were incorporated into IHC diagnostic kits for clinical and research applications. The IHC kits are sensitive and robust for specific detection in FFPE tissues. They hold promise in enhancing cancer diagnosis, identifying patients for targeted therapies, and improving clinical stratification among patients with Claudin family disorders. Citation Format: Qingbo Wang, Xinyue Wang, Jing Tao, Tongyu Xiao, Xiao Wang, Minyang Li, Xiao Lv, Wenwen Zhao, Jiayue Geng, Yixin Liu, Zhanjiao Yu, Yuanhao Li. Development of immunohistochemistry detection kits for claudin family: CLDN6, CLDN9, and CLDN18.2 [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 5914.