CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing
Creators: name: Andersch, Lena
name: Radke, Josefine
name: Klaus, Anika
name: Schwiebert, Silke
name: Winkler, Annika
name: Schumann, Elisa
name: Grunewald, Laura
name: Zirngibl, Felix
name: Flemmig, Carina
name: Jensen, Michael C.
name: Rossig, Claudia
name: Joussen, Antonia
name: Henssen, Anton
name: Eggert, Angelika
name: Schulte, Johannes H.
name: Künkele, Annette
Publisher: name: Charité - Universitätsmedizin Berlin
Year: 2019
Subjects: subject: Adoptive T-cell immunotherapy
subject: Antigen loss
subject: CD171
subject: GD2
subject: Retinoblastoma
subject: Sequential CAR-T cell therapy
subject: 600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit::610 Medizin und Gesundheit

description: BACKGROUND: Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. METHODS: CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. RESULTS: All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. CONCLUSION: Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.