LAUSR

Nanobody-based chimeric antigen receptor (CAR) T cells are currently being tested in early phase clinical trials against blood and solid tumor antigens. Owing to the low molecular weight, increased stability, and low immunogenicity of nanobodies, nanobody-based CAR T cells are an attractive alternative to antibody-based CAR T cells for improving in vivo persistence and overcoming loss of antigen in solid tumors. However, no such agent has been described that can target mesothelin, a tumor antigen that is highly expressed in mesotheliomas, pancreatic, ovarian, and lung carcinomas. Here, a novel nanobody-based CAR T cell that targets both mouse and human mesothelin was created and evaluated in a pre-clinical model of lung adenocarcinoma that encompasses a fully functional murine immune system. To obtain the nanobody, a selection of camel-derived nanobodies were screened for human mesothelin binding using phage panning. Binding of the lead candidate to mouse mesothelin was confirmed using flow cytometry. The antigen binding variable region of the nanobody was incorporated into a second-generation CAR backbone and the sequence was optimized for expression in the mouse host. To generate the nanobody-based CAR T cells, CD3 T cells were isolated from the spleens of naïve mice and transduced using a lentiviral vector. Following expansion, the functionality of the CAR T cells was assessed in cytotoxicity assays and by monitoring the subcutaneous tumor growth rate of the mouse-derived lung adenocarcinoma cell line 344SQ (mouse mesothelin positive) in 129S2/SvPasCrl syngeneic female mice. Murine CAR T cells demonstrated antigen specificity by selectively targeting tumor cells expressing mesothelin in co-culture assays. When mice were treated with a single dose of 10 million CAR T cells, there was a threefold reduction in tumor size one-week post-treatment (n = 6) with complete responses observed approximately two weeks post-treatment in 83% of the treated mice (n = 5/6). Complete responses were maintained for at least two months after treatment in those mice. Collectively, this demonstrates that the nanobody-based CAR T cells can effectively target established subcutaneous tumors even after a single dose and without the need to manipulate the immune system of the host to enhance persistence. This study constitutes a first attempt to establish an immunocompetent pre-clinical model for the study of mesothelin-targeting, nanobody-based CAR T cells in unprimed hosts. This model will facilitate the study of the dynamic changes occurring in the tumor microenvironment following nanobody-based CAR T cell therapy while also providing valuable insight into the tumor resistance mechanisms and methods to overcome them. Citation Format: Chaido Stathopoulou, Jessica Hong, Mitchell Ho, Raffit Hassan. Mesothelin-targeting, nanobody-based CAR T cells effectively target solid tumors in fully immunocompetent hosts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1786.