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CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

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Item Type:Article
Title:CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing
Creators Name:Andersch, L. and Radke, J. and Klaus, A. and Schwiebert, S. and Winkler, A. and Schumann, E. and Grunewald, L. and Zirngibl, F. and Flemmig, C. and Jensen, M.C. and Rossig, C. and Joussen, A. and Henssen, A. and Eggert, A. and Schulte, J.H. and Künkele, A.
Abstract: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.
Keywords:Adoptive T-Cell Immunotherapy, Retinoblastoma, CD171, GD2, Antigen Loss, Sequential CAR-T Cell Therapy
Source:BMC Cancer
Publisher:BioMed Central
Page Range:895
Date:9 September 2019
Official Publication:https://doi.org/10.1186/s12885-019-6131-1
PubMed:View item in PubMed

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