Purpose of study: To determine whether AO-176, a highly differentiated, humanized antibody targeting CD47, shows efficacy alone or in combination with a variety of approved anti-cancer drugs in solid tumors. Methods: We investigated AO-176 as a single agent and in combination with chemotherapies and targeted antibodies, utilizing standard in vitro phagocytosis assays, tumor cell killing assays, and in vivo xenograft models. Results: AO-176 is a highly-differentiated anti-CD47 antibody that not only blocks the CD47/SIRPα interaction to stimulate phagocytosis of tumor cells, but also exerts direct killing activity on tumor cells (non-ADCC), induces immunogenic cell death, and exhibits preferential binding to tumor cells compared to normal cells. In addition, tumor-specific CD47 binding by AO-176 increases in the acidic tumor microenvironment. As a single agent, AO-176 induced cell killing (14-52% Annexin V positivity, EC50 = 1-30 μg/ml) and phagocytosis (10-34%, EC50 = 0.8-3.3 μg/ml) in ovarian, gastric, non-small cell lung, head and neck, colorectal, thyroid, pancreatic and endometrial solid tumor cell lines. When combined with tumor-targeted antibodies (i.e. cetuximab against head and neck and colorectal cancer cell lines, or the checkpoint inhibitor avelumab against ovarian cancer cell lines), AO-176 significantly enhanced phagocytosis of the tumor cells in vitro. In combination with the chemotherapeutics paclitaxel and cisplatin, AO-176 also potentiated direct tumor killing of gastric cancer cells in vitro. In vivo, AO-176 showed potent single-agent anti-tumor activity against ovarian and gastric tumor xenografts. These data add to the previous pre-clinical anti-tumor activity of AO-176 reported in breast cancer, multiple myeloma, and non-Hodgkin’s lymphoma xenografts. When cisplatin or paclitaxel was added to the AO-176 treatment regimen against xenografted ovarian tumors in vivo, significant combination anti-tumor activity was observed. We then sought to extend our promising in-vitro findings from combining AO-176 with a checkpoint inhibitor to in-vivo models. As AO-176 is human CD47-specific, we utilized an in-house murine reactive anti-CD47 blocking antibody and combined it with a murine-reactive anti-PDL1 antibody to treat MC38 murine tumors established in syngeneic mice. Combination of the two antibodies significantly improved anti-tumor efficacy compared to either single agent. Conclusions: AO-176 has demonstrated broad in vitro phagocytosis/killing as well as in vivo efficacy that supports its development, both as a single agent and in combination with other anti-cancer drugs. With a highly differentiated mechanism of action and binding profile, AO-176 may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Citation Format: Casey Wilson, Myriam Bouchlaka, Robyn Puro, Ben Capoccia, Ronald Hiebsch, Prabir Chakraborty, Michael Donio, Vicki Sung, Daniel Pereira. AO-176, a highly differentiated humanized anti-CD47 antibody, exhibits single-agent and combination antitumor efficacy with chemotherapy and targeted antibodies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B100. doi:10.1158/1535-7163.TARG-19-B100
BackgroundCD47 is a cell surface protein expressed on tumors that binds SIRPα on macrophages and dendritic cells resulting in a ”don’t eat me” signal that allows tumors to evade phagocytosis. The highly differentiated monoclonal antibody, AO-176 directly targets CD47 and blocks this signal. AO-176 is currently being tested in phase 1 clinical trials in solid tumors and multiple myeloma. The purpose of this study was to assess in vivo efficacy of AO-176 in solid tumor models as a single agent and in combination with multiple classes of therapeutics including chemotherapeutics, monoclonal antibodies and T-cell checkpoint inhibitors.MethodsCD47 expression levels on solid tumor types were assessed by immunohistochemistry using a tumor tissue microarray. Cell-based binding was performed using flow cytometry under acidic and physiologic pH conditions to characterize the functional activity of AO-176 in the two pH environments representing tumor and normal physiologic environments. In vivo studies were performed using models of solid cancers.ResultsAll 12 solid tumor indications assessed were positive for cell membrane localized CD47 (3.3–98.6 H-scores). Cell-based binding of AO-176 to solid cancer cell lines was significantly greater (1.6–25-fold decrease in EC50, 11–39% increase in Bmax) in acidic conditions as compared to a neutral pH environment, demonstrating improved binding in the lower pH environments associated with solid tumors. AO-176 treatment in solid tumor xenograft models resulted in potent anti-tumor activity as a monotherapy (40–58% TGI) and in combination with paclitaxel in an ovarian model (99% TGI), cisplatin in an ovarian model (84% TGI), cisplatin in a gastric model (76% TGI), and an anti-VEGFR-2 in a gastric model (86% TGI). In vivo efficacy of CD47 blockade alone (~33% TGI) and in combination with anti-PD-1 (74% TGI) and anti-PD-L1 (80% TGI) T-cell checkpoint inhibitors was observed in a syngeneic model of colon cancer using a surrogate anti-CD47 blocking antibody.ConclusionsAO-176 is a differentiated anti-CD47 agent that in addition to blocking the don’t eat me signal, directly kills cancer cells, shows lower binding to normal cells such as RBCs and demonstrates increased binding activity in acidic conditions as found in the microenvironment of solid tumors. AO-176 also elicits potent anti-tumor activity in xenograft and syngeneic models as a single agent and in combination with chemotherapies, monoclonal antibodies and T-cell checkpoint inhibitors. AO-176 is currently in clinical trials as a single agent and in combination in patients with select solid cancers (NCT03834948) and in multiple myeloma (NCT04445701).
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