Background By overexpressing anti-phagocytic surface proteins, often known as "don't eat me" signals, cancer cells can evade macrophage-mediated elimination. Therapeutic antibodies targeting “don't eat me” protein, such as CD47, demonstrates promising anti-tumor efficacy in preclinical models and in clinic. However, the clinical development of anti-CD47 mAbs that retain substantial FcR activating capacity (e.g. human IgG1) has been hampered by the on-target-off-tumor toxicity, such as erythrocyte depletion.1 CD24, a GPI-anchored, highly glycosylated surface protein interacting with Siglec-10 on innate immune cells, was reported to be a novel “don't eat me” protein. CD24 is over-expressed in multiple tumor types. Knock-out of CD24 or blockade of CD24/Siglec-10 interaction enhances macrophage-mediated phagocytosis of tumor cells.2 In this study, we developed a first-in-class, humanized anti-CD24 antibody, ATG-031. The in vitro and in vivo anti-tumor activity of ATG-031 was evaluated in preclinical models.
Methods The affinity of ATG-031 was measured using Surface Plasmon Resonance (SPR). Cell-based binding to HEK293, HEK293-CD24, human red blood cell (hRBC) and a panel of tumor cells was evaluated by flow cytometry. The ability of ATG-031 in enhancing macrophage-mediated phagocytosis of tumor cells was evaluated using human monocyte-derived M2 macrophage. in vivo efficacy of ATG-031 was test in mouse bearing MC38 murine syngeneic colorectal cancer cells stable-expressing human CD24 (MC38-hCD24). Expression profile of CD24 in human tumor and normal tissues were analyzed using TCGA/GTEx database or using an in-house developed companion diagnostic antibody on tissue microarray (TMA) by IHC staining.
Results CD24 is overexpressed in multiple types of solid tumors and hematological malignancies. ATG-031 specifically binds to human CD24 with a single-digit nM affinity. ATG-031 potently binds to CD24-postive tumor cells, while showed no binding with parental HEK293 cells or hRBC. ATG-031 blocks the interaction between CD24 and Siglec-10 and induced potent macrophage-dependent tumor cell phagocytosis (figure 1A, B). Upon phagocytosis, M2 macrophages start to release M1-like cytokines suggesting a repolarization from M2 to M1. ATG-031 significantly inhibited in vivo tumor growth in MC38-hCD24 mouse model. A dose of 3mg/kg ATG-031 administered biweekly induced tumor regression (figure 1C).
Conclusions Blocking CD24 by ATG-031 enhances macrophage-mediated phagocytosis of cancer cells, and polarized M2 macrophage towards anti-tumor M1 subtype (figure 2). It demonstrates potent in vivo anti-tumor tumor efficacy, suggesting promising therapeutic strategies against a broad range of solid tumor or hematological malignancies, which warrants further clinical investigation. A clinical study to investigate the safety and efficacy of ATG-031 in cancer patients is being developed.
Logtenberg MEW, Scheeren FA, Schumacher TN. The CD47-SIRPα Immune Checkpoint. Immunity. 2020 May 19;52(5):742–752
Barkal AA, Brewer RE, Markovic M, Kowarsky M, Barkal SA, Zaro BW, Krishnan V, Hatakeyama J, Dorigo O, Barkal LJ, Weissman IL. CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature. 2019 Aug;572(7769):392–396.
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