D3-GPC2-directed CAR T cells are safe and efficacious in preclinical models of neuroblastoma and small cell lung cancer

Abstract Background: We previously identified glypican 2 (GPC2) as a cell surface MYCN-regulated neuroblastoma oncoprotein and developed a D3-GPC2 antibody that specifically binds a conformational, tumor-specific epitope conserved between mouse and human. Experimental Design: Here we sought to further validate GPC2 as an immunotherapeutic target and develop Investigational New Drug application-enabling data to support the clinical translation of D3-GPC2 chimeric antigen receptor (CAR) T cells. Results: Immunohistochemistry validated that GPC2 is widely expressed on human neuroblastomas and flow cytometry showed high levels of cell surface GPC2 on neuroblastoma cellular models. Second-generation D3-GPC2 CAR T cells with either a 4-1BB or CD28 co-stimulatory domain were selectively activated and induced potent neuroblastoma cell cytotoxicity in several complementary in vitro co-incubation assays. Conversely, no measurable cytotoxicity or D3-GPC2 CAR T cell activation was observed in co-incubation studies with nine primary human normal tissue cell lines. Moreover, GPC2 CAR T cells induced significant regression of GPC2-expressing neuroblastoma xenografts. No GPC2 CAR-related toxicities were noted, including in comprehensive mouse necropsies performed after GPC2 CAR T cell administration. Finally, to explore the potential broader clinical impact of GPC2 CAR T cells we showed that they are also potently cytotoxic to preclinical models of GPC2-expressing small cell lung cancers. Conclusions: These data validate GPC2 as a bona fide CAR T cell target in neuroblastoma and other cancers. The safety and preliminary efficacy of GPC2 CAR T cells are being tested in a first-in-human phase 1 clinical trial for children with relapsed/refractory neuroblastoma (NCT05650749).