CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing
Lena Andersch,
Josefine Radke,
Anika Klaus,
Silke Schwiebert,
Annika Winkler,
Elisa Schumann,
Laura Grunewald,
Felix Zirngibl,
Carina Flemmig,
Michael C. Jensen,
Claudia Rossig,
Antonia Joussen,
Anton Henssen,
Angelika Eggert,
Johannes H. Schulte,
Annette Künkele
Affiliations
Lena Andersch
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Josefine Radke
Department of Neuropathology, Charitéplatz 1, Charité, Universitätsmedizin Berlin
Anika Klaus
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Silke Schwiebert
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Annika Winkler
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Elisa Schumann
Department of Neuropathology, Charitéplatz 1, Charité, Universitätsmedizin Berlin
Laura Grunewald
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Felix Zirngibl
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Carina Flemmig
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Michael C. Jensen
Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute
Claudia Rossig
Department of Pediatric Hematology and Oncology, University Children’s Hospital Muenster, Albert-Schweitzer-Campus 1
Antonia Joussen
Department of Ophthalmology, Charité – Universitätsmedizin Berlin, Hindenburgdamm 30
Anton Henssen
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Angelika Eggert
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Johannes H. Schulte
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Annette Künkele
Department of Pediatric Oncology and Hematology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
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.
