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Background Allogeneic, off-the-shelf CAR T cells offer a potential advantage over autologous CAR T therapy due to their immediate availability, standardized production, and the possibility of repeated dosing and modifications. However, the risk of graft-versus-host disease (GVHD) remains a significant concern. To mitigate GVHD risk, we used healthy donor-derived tumor antigen-specific T cells as source for our CAR T product.We selected diffuse midline glioma (DMG), a highly aggressive and fatal brain tumor with limited treatment options, as our target disease. The use of tumor antigen-specific CAR T cells offers the additional benefit of potentially preventing immune escape, a common challenge in highly heterogeneous tumors including DMG. For this study, we targeted B7-H3, a surface protein, and PRAME, an intracellular protein, both highly expressed by human primary DMG cells.Methods We therefore aimed to characterize the immunobiology of PRAME-specific, B7-H3 CAR T cells engineered to target DMG. To this end, we generated and evaluated PRAME-specific T cells and non-specific T cells, both with and without B7-H3 CAR modifications.Results Our B7-H3 CAR, PRAME-specific T cells showed a mean transduction efficiency of 16.7% and a mean PRAME specificity of 127 IFNγ spots per 100,000 cells (n=4). To demonstrate maintenance of antigen specificity following CAR transduction, we compared PRAME-specific T cells mean IFNγ, TNFα and IFNγ TNFα production in transduced (1.9%) and untransduced (1.9%) and showed comparable specificity.To evaluate whether antigen-specific T cells lack alloreactivity, we tested specificity of tumor-specific T cells compared to their non-specific counterparts with partially HLA mismatched donors. We observed a mean IFNγ spots of 48.6 for PRAME-specific vs 79.6 for non-specific T cells. TCR sequencing of T cells showed a mean 4.6% max productive frequency of tumor-specific CAR modified and unmodified T cells with a respective mean productive Simpson clonality index of 0.08 and 0.07 respectively demonstrating oligoclonal populations as compared to their corresponding non-specific cells (mean 0.3% max productive frequency and Simpson clonality of 0.01).Finally, to evaluate synergistic cytotoxicity from dual targeting, we tested anti-tumor activity against partially-HLA matched DMG cells. We observed enhanced anti-tumor activity in CAR- transduced T cells. However, there was no significant difference in cytotoxicity between antigen-specific and non-specific transduced T cells. This suggests that CAR expression may be saturated on these cells.Conclusions In summary, our results indicate that our platform has potential and may act as attractive and effective combinatorial therapy as an adoptive T cell therapy for solid tumors.