Temporal mapping of the anti-tumor effects of nanobody-based MSLN.CAR-T cell therapy in metastatic solid tumors

Abstract Studies on the dynamic changes occurring in the tumor microenvironment (TME) following CAR-T cell therapy have been confounded by host lymphodepletion, multiple dosing and immunodeficient models. Therefore, CAR-T cell mediated changes in the TME contributing to solid tumor resistance to CAR-T cell therapy remain poorly explored. A nanobody-based, mouse mesothelin-targeting CAR-T cell (A101) was developed to study the changes occurring in the TME after single dose treatment in immunocompetent hosts. Flow cytometry, single cell and total RNA sequencing of tumor tissue were used for TME profiling. A101 CAR-T cells achieved effective primary tumor suppression, metastasis reduction, and improved survival after a single dose in immunocompetent, syngeneic mouse models without lymphodepletion. Temporal tumor profiling using RNA sequencing revealed initial downregulation of cell proliferation genes followed by upregulation of inflammation, epithelial-to-mesenchymal-transition (EMT) and extracellular matrix (ECM) modification genes in the CAR-T-treated tumors relative to mock-T-treated controls. This phenotype was reversed at a later timepoint which coincided with downregulation of immunosuppressive Cd274+ Lcn2+ neutrophils and upregulation of anti-tumor P2rx1+ Nrf2− neutrophils. At the same time, upregulation of Ccl2+ in fibroblasts and a more pro-tumorigenic Ptgs2+ macrophage phenotype was observed in CAR-T-treated tumors, indicating a tumor adaptation mechanism. This study demonstrates complex dynamic changes in the TME, and highlights time-dependent responses of solid tumors to CAR-T cell therapy. It further highlights Ptgs2+ macrophages, Lcn2+ neutrophils and Ccl2+ fibroblasts as potential therapeutic targets for improving CAR-T cell anti-tumor efficacy for solid tumors.