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

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. Here, a nanobody-based, mouse mesothelin-targeting CAR-T cell (A101) was developed, achieving 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 immunomodulatory 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 Lcn2 ⁺ neutrophils and Ccl2 ⁺ fibroblasts as potential therapeutic targets for improving CAR-T cell anti-tumor efficacy.