337 Dual role of CD276 as target antigen and putative activation marker in CD276-redirected CAR T cells for the treatment of solid tumors

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Background CD276 (B7-H3) is an attractive pan-solid tumor target due to frequent overexpression in various solid tumors and tumor stroma, and its correlation with poor clinical outcomes. Despite published CAR T cell therapies targeting CD276 (376.96, MGA271, and B12) and numerous ongoing clinical trials, the therapeutic benefits of these therapies remain underwhelming.1–3 The present study aims to develop novel, superior CAR constructs targeting human CD276 in solid tumors, and to investigate the putative role of CD276 as a T cell activating and modulatory molecule4–7 in the context of CD276-CAR-T cells. Methods CD276-specific CAR binders derived from human scFv and Alpaca libraries were incorporated into a second-generation CAR framework featuring 41BB and CD3ζ signaling domains. Healthy donor T cells were transduced with the CD276-CAR via lentiviral vectors. Cell proliferation, viability, and diameter were closely monitored during product expansion. In vitro assays, including luciferase overnight killing, xCELLigence Real-Time Cell Analysis, and tumor cell rechallenge assays, were performed to evaluate the cytotoxicity of CD276-CAR-T cells against CD276-positive tumor cells. Additionally, the persistence and cytokine secretion profiles of CAR-T cells were assessed. Given CD276’s complex role in T-cell activation, CD276 expression levels were also monitored throughout the process. Results All novel CD276-CAR-T cells showed robust cell expansion and viability, similar to comparator CD276 CARs 376.96 and B12 in vitro. CD276 expression was induced in CD276 CAR-positive CD4+ and CD8+ T cell products construct-dependently. In overnight killing assays, all CD276-CAR-T cells mounted IFN-γ, IL-2, and TNF-alpha cytokine response, and effectively and specifically killed CD276-positive ovarian, pancreatic, and lung tumor cell lines (OVCAR-3, AsPC-1, NCI-H226), with no impact on CD276-negative cells (RS4.11). By contrast, in a long-term tumor cell rechallenge assay, novel CARs D0506 and D0537, which exhibited higher CD276 expression in cell product and upon antigen stimulation, also tended to produce strong target-dependent expansion, persistence and serial tumor cell killing. Conclusions The novel CD276-CAR-T cells CARs D0506 and D0537 maintained persistence and potent cytotoxicity upon long-term tumor cell re-challenge, concordantly with heightened CD276 surface expression during manufacturing and target-dependent activation. These findings point to CD276 as a putative activation marker and modulator of CAR-T cell function, and may inform further CD276 CAR engineering strategies. References Vitanza NA, Wilson AL, Huang W, et al. Intraventricular B7-H3 CAR T cells for diffuse intrinsic pontine glioma: preliminary first-in-human bioactivity and safety. Cancer Discovery 2023;13(1):114–31. Pinto NR, Albert CM, Taylor M, et al. STRIVE-02: a first-in-human phase 1 trial of systemic B7H3 CAR T cells for children and young adults with relapsed/refractory solid tumors. Journal of Clinical Oncology 2022;40(16_suppl):10011–11. Epperly R, Gottschalk S, DeRenzo C. CAR T cells redirected to B7-H3 for pediatric solid tumors: current status and future perspectives. EJC Paediatric Oncology 2024;3:100160. Picarda E, Ohaegbulam KC, Zang X. Molecular pathways: targeting B7-H3 (CD276) for human cancer immunotherapy. Clinical Cancer Research 2016;22(14):3425–31. Chapoval AI, Ni J, Lau JS, et al. B7-H3: A costimulatory molecule for T cell activation and IFN-γ production. Nature Immunology 2001;2(3):269–74. Riccione KA, Gedeon P, Sanchez-Perez L, et al. Chapter 11 - Checkpoint Blockade Immunotherapy for Glioblastoma: Progress and Challenges. In: Sampson JH, ed. Translational Immunotherapy of Brain Tumors. San Diego: Academic Press, 2017:261–300. Zhang G, Hou J, Shi J, et al. Soluble CD276 (B7-H3) is released from monocytes, dendritic cells and activated T cells and is detectable in normal human serum. Immunology 2008;123(4):538–46.

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