Figure 3. Killing by B7-H3 nanoCAR-T cells is antigen-specific. (A) Graphical representation of CAR-T cell killing of GFP^pos B7-H3^pos LN229 glioblastoma cells but not Katushka2S^pos B7-H3^neg LN229 cells. (B) B7-H3 expression on wild type LN229 cells and B7-H3 knock-out LN229 cells. (C) GFP expression in wild type LN229 cells and Katushka2S expression in B7-H3 knock-out LN229 cells, following stable transduction with lentiviral vectors. (D) Live cell images from IncuCyte showing killing of GFP^pos target cells but not Katushka2S^pos non-target cells in three-dimensional tumor models by CAR-T cells, as shown for one representative donor. (E) End-point flow cytometric analysis of % target cell specific killing. Data represent mean±SD n=3, biological repeats. Each dot represents a different donor. *p≤0.05. Statistical significance in panel E was determined using an unpaired t-test. 5T2Id, 5T2 multiple myeloma idiotype; B7-H3, B7 homolog 3; CAR, chimeric antigen receptor; nanoCAR, nanobody-based chimeric antigen receptor; scFvCAR, single-chain variable fragment-based chimeric antigen receptor.