Antibody-mediated CAR T Platform for Targeting Tumor Cells and the Tumor Microenvironment 2301697

Abstract Introduction CAR T cell therapy has shown success in treating hematologic malignancies but faces challenges in solid tumors due to antigen heterogeneity and the complex tumor microenvironment (TME). CAFs expressing fibroblast activation protein (FAP) contribute to immune suppression by creating physical barriers and immune-suppressive environment that blocks T cell infiltration and persistence. Overcoming these barriers is crucial for enhancing CAR T cell therapy in solid tumors. Methods We engineered a modular CAR T cell platform using anti-FLAG CAR T cells conjugated with FLAG-tagged monoclonal antibodies, allowing flexible targeting of both tumor cells and CAFs. This platform was tested in B-cell lymphoma and pancreatic cancer models, both in vitro and in vivo. Cytotoxicity was evaluated through flow cytometry. TME remodeling and cytokine production were assessed using immunostaining, multiplex cytokine assays, and qPCR. In vivo efficacy was evaluated in subcutaneous and orthotopic models. Results In B-cell lymphoma, FLAG CAR T cells, combined with FLAG-tagged CD19 antibodies, exhibited strong anti-tumor activity, significantly increasing tumor cell lysis and achieving cytotoxicity comparable to traditional CD19 CAR T cell therapies. In solid tumor models, FLAG CAR T cells, in combination with FLAG-tagged antibodies targeting FAP+ CAFs and Claudin18.2+ tumor cells, enhanced immune cell infiltration and significantly suppressed tumor growth. This dual-targeting strategy disrupted the TME-induced immune suppression, reduced collagen deposition, and boosted T cell activity, resulting in notable tumor growth inhibition and prolonged survival in both subcutaneous and orthotopic pancreatic cancer models. Conclusion This CAR T cell platform enables dual targeting of tumor cells and CAFs, overcoming immune suppression in solid tumors. This approach enhances immune activation, reshapes the TME, and provides a promising strategy for improving CAR T cell therapy in solid tumors. Funding Source This study was funded by the National Natural Foundation of China (No. 81970632), Guangdong Science and Technology Department (Nos. 2020B1212060018 and 2020B1212030004), Guangdong Basic and Applied Basic Research Foundation 2025A1515011234. Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)