CAR T-cell therapy against the immune checkpoint B7-H3 in patient-derived 3D-models from Head and Neck Cancer (HNC)

Background:So far, CAR T-cell therapy has not been successful in solid tumors, due to several factors including heterogeneous expression of target antigens and a hostile tumor microenvironment. The immune checkpoint molecule B7-H3 is highly expressed on solid cancers and cancer-associated fibroblasts, but hardly detectable on healthy tissue, and thus a promising target for immunotherapy.

Methods:The expression of B7-H3 was analyzed on 24 HNC cell lines by flow cytometry. The cytotoxicity and specificity of human T-cells expressing three B7-H3 CAR constructs was evaluated against HNC cells in MTT assays. B7-H3 knockout cells were created by a lentiviral CRISPR/Cas9 to detect off-target effects of B7-H3 CARs. For 3D in vitro systems, patient-derived organoids (PDOs) were established from fresh HNC tumor samples and spheroids from HNC cell lines.

Results:All HNC cell lines expressed B7-H3 at high levels and were efficiently and specifically killed by B7-H3 CAR T-cells. PDOs were successfully established in ~40% of specimen and cultured over multiple passages. Analysis of PDOs after 3-4 weeks revealed the presence of B7-H3-positive malignant cells and cancer-associated fibroblasts, while immune cells were largely absent. While the cytotoxicity of B7-H3 CAR T-cells in PDOs was technically challenging to detect, selective CAR T-cell killing of mixed spheroids containing B7-H3 positive and negative HNC cells was readily established using flow cytometry and live-cell imaging.

Conclusion:B7-H3 is an excellent target antigen for CAR T-cell therapy in HNC targeting malignant as well as non-malignant cells. HNC cell lines-derived spheroids and PDOs can readily be established as promising in vitro models for addressing current obstacles in the CAR immunotherapy of solid tumors.

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