Abstract 2862: Anti MSLN ADC RC88 engages unique binding to target positive tumor cells with less interference from the soluble MSLN protein

Abstract Background: Mesothelin (MSLN) is a cell surface protein in various cancers, including ovarian, pancreatic and malignant mesothelioma. Soluble mesothelin (sMSLN) can be detected in the circulation of patients and may reduce the efficacy of antibody drugs targeting surface MSLN. Acting as a decoy receptor, sMSLN competes for binding or neutralizes the drug, affecting antibody properties. This study examined the anti MSLN antibody-drug conjugate (ADC) RC88, focusing on its ability to target cancer cells and the interference caused by sMSLN, thereby highlighting its competitive advantage over other drugs in clinical studies. Methods: RC88, a novel anti MSLN ADC developed by RemeGen, is in phase I/II trials for advanced solid tumors, either as monotherapy (NCT04175847, NCT06173037) or in combination therapy (NCT05804526). We compared RC88 with other anti MSLN antibodies from relevant trials by antigen and cell binding assays. To show the unique binding of RC88, we treated cellular MSLNs in various conditions to mimic in vivo pathological environments. Additionally, we expressed a truncated MSLN fragment in 293 cells to focus on the C-terminal region near the cell membrane for binding comparisons. Results: Our analysis revealed the distinctive properties of RC88 that may provide clinical advantages. RC88 had a strong primary binding site in the N-terminal domain and a weaker secondary site in the C-terminal region. The primary binding site at amino acids 317-352 was characterized by cross-linking mass spectrometry and mutagenesis. This epitope also facilitates CA125 binding, and RC88 effectively blocks CA125 from binding to MSLN. The secondary binding site located in the C-terminal stem region (amino acids 577-606) was less prominent and appeared to enhance tumor cell binding of RC88. Although further studies are required to refine the location of this secondary site, RC88 demonstrated its ability to bind and induce ADC-mediated cell death in cells expressing the C-terminal fragment of MSLN, a response not seen with other anti MSLN antibodies. When comparing cell binding in the presence of sMSLN, RC88 showed stronger cancer cell binding than others. Conclusions: Our findings show RC88 has unique binding kinetics to different MSLN regions, with a dominant N-terminal binding site by rigorous methods. RC88 displayed superior binding to MSLN-positive cancer cells than other anti MSLN antibodies and maintained effectiveness despite sMSLN interference. Its stronger binding to the C-terminal MSLN fragment compared with other antibodies further underscores its targeting potential in tumor cells. Given its distinctive binding properties, RC88 may bring enhanced clinical benefits for treating MSLN-positive cancers, even with sMSLN and various truncated forms of MSLN in tumor cells. Therefore, RC88 is likely to have greater clinical benefits than other MSLN ADCs in clinical studies. Citation Format: Lili Wang, Yidan Xu, Mingyang Li, Kailin Wang, Xiao Wang, Xiaoping Zhang, Zhanjiao Yu, Yinghao Xin, Chenglin He, Xiangyi He, Xiaoshan Min, Hang Chen, Min Li, Zhulun Wang, Dong Li, Yuanhao Li. Anti MSLN ADC RC88 engages unique binding to target positive tumor cells with less interference from the soluble MSLN protein [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 2862.