Figure 7. Reactivity of B7-H3 nanoCAR-T cells in non-tumor-bearing mice confirm the occurrence of on-target, off-tumor toxicity. (A) Graphical overview of in vivo experimental set-up. (B) Flow cytometry data of blood from mice, processed at 3, 4 and 5 weeks of CAR-T cell treatment, showing % of circulating human CD45 cells gated within all living blood cells. (C) Weight curves over time. (D) Hematological analysis of blood after 5 weeks of B7-H3 nanoCAR or HER2-CAR therapy evaluating red blood cell, hemoglobin and hematocrit levels as determined by the scil Vet abc Plus. (E) Images of spleens dissected from mice after 5 weeks of B7-H3 nanoCAR or HER2-CAR therapy. (F) Flow cytometry data of spleens from mice, processed at 5 weeks of CAR-T cell treatment, showing % of human and mouse CD45 cells gated within all living spleen cells (G) Immunohistochemical staining of spleens from the indicated mice, assessing CAR-T infiltration based on human CD3 staining. (H) Flow cytometry data of bone marrow cells from mice, processed at 5 weeks of CAR-T cell treatment, showing % of human and mouse CD45 cells gated within all living bone marrow cells. (I) Immunohistochemical staining of femurs from the indicated mice, assessing CAR-T infiltration based on human CD3 staining. (J) Representative Immunohistochemical staining of brain, heart, lung, liver, small intestine and kidney of mice with human CD3 antibody to detect CAR-T infiltration. Data represent mean±SD n=3 mice per group. B7-H3, B7 homolog 3; CAR, chimeric antigen receptor; HER2, human epidermal growth factor receptor 2; i.v., intravenously; nanoCAR, nanobody-based chimeric antigen receptor.