Exth-80. a systemic comparison of car t cells, car nk cells and car macrophages for glioma treatment

Adoptive transfer of engineered immune cells expressing chimeric antigen receptors (CAR) is an emerging cancer immunotherapy that is also being investigated for glioblastoma. Many separate studies focus on either CAR T or CAR NK cells, and CAR macrophages are in early stages of development. However, a systemic cross-comparison of engineered immune effector cells against solid tumors, including glioblastoma, is lacking. Here, we generated second-generation NKG2D CAR mouse and human T cells, NK cells and macrophages and characterized them in vitro in co-culture assays with glioma cells and in vivo in orthotopic glioma mouse models. In vitro, mouse and human CAR T cells and CAR NK cells exhibited the highest tumor-killing activity. In contrast, in vivo, only CAR T cells exhibited anti-tumor activity in two immunocompetent orthotopic glioma-bearing mouse models, whereas CAR NK cells and CAR macrophages failed to prolong survival. More in-depth characterizations using flow cytometry, in vivo imaging and 3D microscopy, revealed that CAR T cells had the best tumor migration properties upon i.v. administration with distinct spatial distribution within the tumor compared to CAR NK cells and CAR macrophages. To profile the effects on bystander immune cells within the tumor microenvironment, we employed flow cytometry and single-cell RNA sequencing. This revealed effector cell-specific effects on the tumor microenvironment and provided insights into why the effective in vitro activity did not translate to in vivo. To unleash the anti-tumor potential of CAR immune effector cells, we engineered them to co-express the pro-inflammatory cytokines IL-12/IFNα2. This improved the in vivo anti-tumor activity of all effector cells and led to the highest fraction of long-term surviving mice with CAR NK cells. This study offers the first cross-comparison of different CAR immune effector cells against gliomas, providing valuable insights for the design of future adoptive cell therapies targeting malignant brain tumors.