Abstract 43: Targeting trogocytosis through cathepsin B inhibition enhances CAR T cell persistence
Authors: Dietze Kenneth A. , Olson Michael L. , Gebru Etse , Atanackovic Djordje , Rapoport Aaron P. , Luetkens Tim 
Published: March 2024

Abstract
Chimeric Antigen Receptor (CAR) T cell therapy is an effective treatment for cancer patients. While CAR T cells have shown remarkable efficacy, most patients receiving CAR T cell therapy eventually relapse. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism involving the transfer of cell surface proteins from tumor cells to CAR T cells. CMT results in antigen-negative tumor cells and correlates with reduced CAR T cell persistence, possibly due to increased CAR T cell fratricide or exhaustion. To date it has not been conclusively demonstrated that antigens transferred by CMT are the cause of fratricide or exhaustion. To answer this question, we developed a system to rapidly degrade trogocytosed protein in CAR T cells. Using this system, we show that the transfer of CD19 to CAR T cells directly causes CAR T cell fratricide and exhaustion. We hypothesize that reducing CMT will increase CAR T cell persistence. To identify molecular mechanisms driving CMT as potential therapeutic targets, we inhibited various proteins essential for cell adhesion, endocytosis, actin polymerization, or antigen processing using small molecule inhibitors. We found that inhibition of the cysteine protease Cathepsin B (CTSB), with a membrane-permeable (Ca-074) or membrane-impermeable (Ca-074-Me) inhibitor, significantly reduced CMT without affecting CAR T cell cytotoxicity. We hypothesize that CTSB inhibition could be a potential therapeutic approach to limit CMT.CTSB activity is regulated by the protein Cystatin A (CSTA). Both Ca-074-Me and CSTA sterically block access to the active site cysteine of CTSB. We found that overexpression of CSTA in CAR T cells significantly reduced CTSB activity and reduced antigen transfer. CSTA overexpression minimized antigen loss on tumor cells, indicating that CTSB inhibition blocks CMT early during the trogocytic process. CSTA overexpression reduced CAR T cell exhaustion but did not alter their expansion, tumor cell killing, or phenotype, indicating that this approach selectively inhibits CMT. Next, we assessed the effect of CSTA overexpression on CAR T cell persistence in vivo. NALM6 tumor-bearing NSG mice were injected with conventional or CSTA-overexpressing CD19 CAR T cells. Four weeks after CAR T cell injection, we observed substantially increased CAR T cell numbers in the blood (10.6x, p=0.0317), bone marrow (7.3x, p=0.0159), and spleens (5.6x, p=0.0556) of mice treated with CSTA-overexpressing CAR T cells. Taken together, we provide the first experimental evidence that CMT directly causes fratricide and exhaustion, reducing CAR T cell persistence. We demonstrate that CMT can be targeted efficiently by overexpressing the CTSB inhibitor CSTA in CAR T cells, resulting in substantially increased CAR T cell persistence. This represents a promising approach to improve CAR T cell efficacy and limit the occurrence of relapse in patients receiving CAR T cell therapy.
Citation Format: Kenneth A. Dietze, Michael L. Olson, Etse Gebru, Djordje Atanackovic, Aaron P. Rapoport, Tim Luetkens. Targeting trogocytosis through cathepsin B inhibition enhances CAR T cell persistence [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 43.