Development of GD2- and FAP- targeted CAR T-cell strategies to overcome tumor and stroma barriers in osteosarcoma

Osteosarcoma (OS) is one of the most aggressive pediatric malignancies, characterised by limited survival rates and poor outcomes in cases of relapsed or metastatic disease. Although chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic malignancies, its application to solid tumors has been hampered by antigen heterogeneity and the immunosuppressive tumour microenvironment (TME). Within this hostile niche, the stromal compartment, especially cancer associated fibroblasts (CAFs), plays a pivotal role in establishing a dense stromal network that restricts immune cell infiltration, remodels the extracellular matrix, and impairs T-cell recruitment and activation. In this preclinical study, we developed a dual-target CAR T-cell strategy to overcome stromal-mediated CAR T-cells resistance and enhance anti-tumor efficacy in OS models. In particular, we engineered CAR T-cells directed against the disialoganglioside GD2, a tumor associated antigen highly expressed in OS, and against the fibroblast activation protein (FAP), a type II transmembrane serine protease selectively expressed by activated fibroblasts and OS cells, but typically absent in most normal adult tissues. Transcriptomic analyses of RNA-seq datasets from the St. Jude Cloud–Pediatric Cancer Data Portal confirmed high expression of the GD2-synthase enzyme B4GALNT1 across pediatric solid tumors, particularly in OS, and cytometric analyses identified the disialoganglioside GD2 as a robust and clinically relevant tumor-associated antigen, expressed in over half of primary OS samples and upregulated in established cell lines. Third-generation GD2.CAR T-cells, based on a clinically available construct currently under evaluation for pediatric neuroblastoma (NCT03373097), efficiently eradicated GD2+ OS cells in 2D/3D co-culture models (p