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Background B7-H3 (CD276), a member of the B7 family, is a compelling therapeutic target due to its selective expression on tumor cells and immune cells within the tumor microenvironment. 1 Our previous work identified CD276 as an activation marker in CD276-CAR-T cells2; however, its functional role in CAR-T biology remains poorly understood. Here, we examine the impact of CD276 expression on the anti-tumor efficacy of CD276 CAR-T cell products.Methods CD276-binding moieties were generated from a human phage display library (scFv) or an immunized alpaca (nanobody) B cell library with CD8α hinge/transmembrane domains, and intracellular 4-1BB and CD3ζ signaling domains. CARs targeting ROR1, CD19, MSLN, and BCMA were tested to determine whether CD276 upregulation is a feature of CAR-T cell products. CD276 expression in healthy human T cells and CAR-T cells was evaluated using anti-CD276 antibody (Miltenyi Biotec, REA1094/FM276). Fratricide was assessed by fluorescently labeling target cells to distinguish them from effector CAR-T cells. The contribution of trogocytosis was determined using Latrunculin A inhibition. Endogenous CD276 was knocked out using two specific CRISPR-Cas9 constructs (here, #1 and #2) in primary human CAR-T cells. Functional evaluations included proliferation assays, luciferase-based cytotoxicity, and serial killing assays.Results CD276 expression was upregulated during T cell expansion, independent of IL-2 or IL-7/IL-15 culture conditions or the type of CAR expressed (CD276-, ROR1-, MSLN-, BCMA-, or CD19). Increased CD276 expression was observed upon tumor engagement, correlating with T cell activation. Direct fratricide by CD276 CAR-T cells was demonstrated by the killing of activated (but non-CAR expressing) labeled CD276+ T cells. Importantly, trogocytosis played a role in limiting CAR-T fitness and was associated with impaired expansion. CRISPR-mediated CD276 knockout significantly reduced CD276 expression and rescued a high proportion of CD4+ T cells during expansion, resulting in enhanced proliferation and a CD4-enriched final product. While luciferase-based killing assays showed minimal differences, CD276 knockout (#1 and #2) substantially improved serial killing capacity and sustained CAR-T cell proliferation.Conclusions CD276 upregulation is a common consequence of T cell and CAR-T cell activation. In CD276-CAR-T cell populations, this activation-induced expression may contribute to fratricide by increasing the target antigen, thus limiting expansion and functional persistence. T cell impairment is driven by both trogocytosis and endogenous target antigen stimulation. Knockout of CD276 reverses these effects, supports enhanced proliferation, and improves cytotoxicity. These findings suggest that targeting endogenous CD276 is a viable strategy for optimizing CD276-CAR-T cell therapies and improving clinical outcomes.References Picarda E, Ohaegbulam KC, Zang X. Molecular pathways: targeting B7-H3 (CD276) for human cancer immunotherapy. Clinical Cancer Research 2016;22(14):3425-31. doi: 10.1158/1078-0432.CCR-15-2428Xiong W, Hu P, Wagner M, et al. 337 dual role of CD276 as target antigen and putative activation marker in CD276-redirected CAR T cells for the treatment of solid tumors. Journal for Immunotherapy of Cancer 2024;12(Suppl 2):A389. doi: 10.1136/jitc-2024-SITC2024.0337