Abstract 1032: Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies
Authors: Chen Jishun , Bai Suya , Bai Yu , Hu Zaoshun , Chen Peng , Mei Jay , Shan Bo , Hou Bing 
Published: March 2024

Abstract
Background: CLDN18.2, the isoform 2 of CLDN18, is physiologically confined to gastric mucosa tight junctions, the epitopes of which would be exposed on the cancer cell surface upon malignant transformation and is highly expressed in a significant proportion of cancers, e.g., gastric and pancreatic adenocarcinomas, which makes it a promising drug target for cancer therapy. Currently, multiple drug modalities, such as monoclonal antibodies, ADCs, and CARTs targeting CLDN18.2 are being evaluated in clinical trials. A substantial correlation exists between the expression of CLDN18.2 and the efficacy of the these therapies. However, to date, no companion diagnostic (CDx) antibodies that are specific to CLDN18.2 have been approved. Therefore, it is important to promptly develop an anti-CLDN18.2 CDx antibody with a high specificity, sensitivity, and suitability for immunohistochemistry (IHC). In this study, we present the discovery and validation of a novel, highly sensitive IHC antibody that selectively identifies CLDN18.2.
Methods: 3 CLDN18.2 specific peptide fragments from extracellular domain of human CLDN18.2 were conjugated with KLH. Antibodies were discovered by immunizing BALB/c and SJL mice with these peptides-KLH conjugates. Screening and selection process was relied on positive ELISA results against the immunogen, as well as FACS and Acumen Explorer Microplate staining of CLDN18.2-experssing cells. Hit antibodies were evaluated for IHC on formalin-fixed paraffin-embedded CHOK1 cells expressing full-length CLDN18.2 or CLDN18.1. The antibodies were further validated on human gastric cancer tissue samples to establish the analytical sensitivity and specificity of the IHC assay.
Results: The mAb clone 43F11 exhibited binding to a fusion polypeptide comprising the first extracellular loop of human CLDN18.2 (aa32-53) at a half maximal effective concentration (EC50) of 0.212 nM. 43F11 showed positive cell surface IHC staining on CLDN18.2-experssing cells after fixation but demonstrated no staining on CLDN18.1-experssing cells. The IHC test method was established by utilizing clinical tumor tissues and PDX samples that had predetermined expression levels of CLDN18.2. The 43F11 antibody accurately identified the expression level of CLDN18.2 in the IHC assay, with a minimum concentration of 0.25 μg/mL. When compared to the commercial CLDN18.2 IHC antibody EPR19202, 43F11 demonstrated greater sensitivity, allowing for positive staining on cancer tissues that have significantly lower levels of CLDN18.2 expression.
Conclusion: 43F11 exhibits specific recognition of human CLDN18.2 in IHC assays, while showing no reactivity towards CLDN18.1. It demonstrated superior sensitivity compared to the benchmark antibody. These data suggest that 43F11 has the capacity to serve as an effective diagnostic tool for identifying patients who positively express CLDN18.2.
Citation Format: Jishun Chen, Suya Bai, Yu Bai, Zaoshun Hu, Peng Chen, Jay Mei, Bo Shan, Bing Hou. Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1032.