Abstract 41: Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning

Abstract Purpose: Mesothelin (MSLN) is a surface antigen associated with tumor invasion and is overexpressed in mesothelioma and lung, pancreatic, ovarian, and other cancers. Chimeric antigen receptor (CAR) T cells targeting MSLN have been tested in patients with MSLN-positive solid tumors. Despite reported patient fatalities in trials sponsored by PENN, Memorial Sloan Kettering/Atara Bio, and MD Anderson/TCR2, the design approach for MSLN CAR remains constructing high affinity CARs to maximize target cell killing. In this study, we designed MSLN CARs with two different antibodies: one targeting human MSLN specifically and another binding both human and mouse MLSN with comparable affinities. The objectives are to 1) elucidate the risk of fatal on-target off-tumor toxicity associated with high affinity MSLN CAR, and 2) demonstrate how optimizing the affinity of CARs can render CAR T cells to spare normal tissues while retaining cytotoxicity against tumors. Methods: To asses on-target off-tumor toxicity in animal models, we developed a CAR using a nanobody isolated from an immune alpaca library with nanomolar affinity for both human and mouse MSLN (labeled as hmMSLN CAR). For comparison, we constructed a CAR using a scFv specific only to human MSLN, designated hMSLN CAR. Each CAR is designed to co-express somatostatin receptor 2 to enable PET/CT imaging of CAR T cells with 18F-NOTA-Octreotide. Anti-tumor activities and toxicities were assessed in NSG mice using MSTO-211H cell line expressing human MSLN. Results: In NSG mice bearing subcutaneous MSTO-211H tumors, hMSLN CAR T cells eliminated tumors rapidly without inducing any overt toxicity. In contrast, hmMSLN CAR T cells not only failed to control tumor growth but also caused severe toxicity, resulting in significant body weight loss and necessitating euthanasia. PET/CT imaging revealed while hMSLN CAR T cells expanded exclusively within the tumor and subsequently contracted after tumor elimination, hmMSLN CAR T cells exhibited extensive infiltration and expansion in the lungs, liver, and bone marrow, indicating off-tumor, normal tissue-driven proliferation of CAR T cells. To test if affinity-tuned hmMSLN CAR T cells can selectively target tumors, we used a yeast display system to screen for affinity variants of the hmMSLN CAR. We identified one micromolar affinity CAR that binds approximately 1,000 times weaker to MSLN than the parental CAR. In stark contrast to the onset of severe toxicity soon after the administration of the parental hmMSLN CAR T cells, animals treated with micromolar affinity hmMSLN CAR T cells exhibited tumor regression without overt toxicity. Conclusions: Our findings underscore the risks of using high affinity CAR against MSLN, which may cause unpredictable on-target, off-tumor toxicities. Fine tuning the affinity of MSLN CAR offers a promising strategy for developing a potent and safe CAR for the treatment of MSLN-expressing solid tumors. Citation Format: Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Ju Y. Son, Niloufar Salehi, Irene M. Min, Moonsoo M. Jin. Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning [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 41.