Abstract 2996: Internalization of B7-H3 is enhanced by cholesterol disruption in esophageal cancer cells

Abstract B7-H3, a member of the B7-family protein, is highly expressed on cancer cells. An antibody-drug conjugate (ADC) targeting B7-H3 showed potential efficacy in early-phase clinical trials. As endocytosis and subsequent degradation of the ADC drugs are the key processes to release the payload and demonstrate cellular toxicity, alteration in the endocytosis pathway could play a role in the resistance mechanisms or synergistic therapeutic effects. As for HER2-targeted ADCs, one of the most developed ADC targets, some preclinical trials reported a synergistic effect of HER2-targeted ADCs and modulators on the dynamics of HER2 on the cellular membrane, such as membrane cholesterol and HSP90. However, such machinery for B7-H3 and the targeting antibody remains unclear. Thus, we hypothesized that modulating the stability of the membrane B7-H3 could have a synergistic effect with B7-H3 ADC. Using flow cytometry, we evaluated the decrease of surface expression of the targeted antigens as a surrogate marker of their internalization efficacy in TE4, an esophageal squamous cell carcinoma cell line, which showed high expression of both B7-H3 and HER2. Trastuzumab and enoblituzumab, anti-HER2 and -B7-H3 antibodies, respectively, were used to induce endocytosis of target antigens. We validated that there was no epitope interference between therapeutic antibodies, trastuzumab or enoblituzumab, and fluorescent-labelled antibody to detect HER2 or B7-H3. Internalization efficacy was evaluated by comparing surface expression of HER2/B7-H3, between trastuzumab/enoblituzumab and an isotype antibody. The stability at the membrane was modulated by two drugs, pimitespib, an inhibitor of HSP90, and methyl-beta-cyclodextrin (MbCD), a cholesterol disruptor. In flow cytometry analysis, neither pimitespib nor MbCD did not affect the cell surface expression of HER2 and B7-H3. While the pimitespib enhanced trastuzumab-mediated HER2 internalization, enoblituzumab-mediated B7-H3 internalization was not affected. Under MbCD treatment, surface expression of both HER2 and B7-H3 was markedly decreased after administration of targeted antibodies. Additionally, fluorescent immunocytochemistry showed the enhanced internalization of HER2 and B7-H3 molecules at the cell membrane into the cytoplasm. These results suggest that membrane cholesterol, rather than HSP90, might affect the stability of B7-H3 on the cell membrane. In conclusion, membrane cholesterol disruption could enhance the internalization efficacy of HER2 and B7-H3 molecules. Citation Format: Takashi Imajima, Kenji Tsuchihashi, Takafumi Kitazono, Wataru Kusano, Eishi Baba. Internalization of B7-H3 is enhanced by cholesterol disruption in esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2996.