Background CD24 is a small, highly glycosylated cell adhesion protein that functions as a novel ‘don’t eat me’ target in cancer, where it is the dominant innate immune checkpoint.1 Through its interaction with Siglec-10 expressed by tumor-associated macrophages, overexpression of CD24 on tumor cells prevents them from being engulfed by macrophages. Therapeutic antibodies blocking ‘don’t eat me’ signals exhibited potent preclinical and early clinical efficacy against solid tumor and hematological malignancies.2 ATG-031 is the first humanized anti-CD24 monoclonal antibody that enters clinic. Here, we present the ATG-031 preclinical results, including the pharmacology, pharmacodynamics (PD), pharmacokinetics (PK), and safety profiles.
Methods The expression profile of CD24 was evaluated using IHC on a tissue microarray. ATG-031 underwent in vitro pharmacology assessments including but not limited to binding affinity, phagocytosis, receptor blockage, ex vivo cytokine release, and ADCC and CDC assays. In multiple syngeneic models, the in vivo efficacy of ATG-031 was evaluated. PD of ATG-031 was investigated using flow cytometry and single cell RNA sequencing of TILs isolated from MC38-hCD24 tumor samples following ATG-031 administration. ATG-031 and ATG-031-CM, a cynomolgus monkey surrogate antibody, were utilized for toxicology and PK studies in non-human primates (NHP).
Results CD24 is extensively expressed in a variety of tumor types, whereas its distribution in normal tissues is limited (figure 1). ATG-031 binds to the protein backbone epitope of CD24 with affinity in the nM range against CD24 protein or tumor cells. With an EC50 in the sub-nM range, ATG-031 induces potent macrophage-dependent tumor cell phagocytosis. Phagocytosis induced by ATG-031 occurred within 5–10 minutes of incubation based on confocal imaging of live cells (figure 2). M2 macrophages begin to release M1-like cytokines upon phagocytosis. With an EC50 of 0.27nM, ATG-031 induced a potent ADCC effect against HT-29 tumor cells. ATG-031 exhibited potent antitumor activity in vivo as a single agent in syngeneic models such as MC38, CT26, and 4T1. It also demonstrated in vivo synergism with chemotherapies and checkpoint inhibitors. Treatment with ATG-031 significantly increased T cell-associated immune response at the cellular and single-cell transcriptome levels (figure 3). ATG-031 and ATG-031-CM were well tolerated in NHP, with toxicities consistent with the mechanism of action and target expression.
Conclusions In preclinical studies, ATG-031 demonstrates potent antitumor activity, excellent safety and PK properties, which supports its further development in clinical trials. A phase I, multicentre, dose-escalating clinical trial is being developed to evaluate ATG-031 in patients with solid tumors and hematologic malignancies.
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.
Li W, Wang F, Guo R, Bian Z, Song Y. Targeting macrophages in hematological malignancies: recent advances and future directions. J Hematol Oncol. 2022 Aug 17;15(1):110.
Ethics Approval The protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the IACUC of CrownBio. All studies were conducted following an approved IACUC protocol. AUP NO.:2004–12-1465, 2004–12-1000; IACUC approval number: IACUC-2021-M-003
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