Article Text

Download PDFPDF

1337 The preclinical characterization and translational research of ATG-031, a first-in-class humanized anti-CD24 antibody, for the treatment of solid tumors and hematological malignancies
  1. Peng Chen1,
  2. Jishun Chen1,
  3. Min Deng1,
  4. Jiamei Luo1,
  5. Yun Liu1,
  6. Rong Guo1,
  7. Yunkai Zhang2,
  8. Linjie Tian2,
  9. Jay Mei1,
  10. Bo Shan1 and
  11. Bing Hou1
  1. 1Antengene Corporation Limited, Shanghai, China
  2. 2Antengene Biotech LLC, Doylestown, PA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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.


  1. 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.

  2. 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

Abstract 1337 Figure 1

The expression profile of CD24. (A) CD24 showed much higher tumor expression (TCGA) and narrower normal tissue distribution (GTEx), with significantly lower normal heart and CNS expression, compared with CD47. (B) A highly selective CDx antibody for IHC staining was developed in house. IHC on tumor tissue microarray revealed that 50-80% of patients with lung, breast, bladder, ovarian, or liver cancer have CD24 expression on tumor cell surface.

Abstract 1337 Figure 2

ATG-031 induces potent macrophage dependent phagocytosis of tumor cells. (A-B) Live-cell, time-lapse confocal imaging of human monocyte derived M2 macrophage (green) co-cultured with Nalm-6 leukemia cells (red), treated with ATG-031 or IgG control. ATV-031 induces potent macrophage-dependent tumor cell phagocytosis. The imaging duration was 30 minutes (A) and 20 hours (B), respectively. Phagocytosis induced by ATG-031 occurred within 5-10 minutes of incubation. The engulfed Nalm-6 were completely digested by macrophages within 10 hours (B) IgG isotype control did not induce phagocytosis. The videos will be included in the presentation. (C) The macrophage phagocytic index against tumor cells induced by ATG-031, benchmark anti-CD24 antibody (SN3) or IgG1 control.

Abstract 1337 Figure 3

(A) C57BL/6 mice bearing MC38-hCD24 tumors were treated twice with 3 mg/kg ATG-031 or IgG1 control (Arrow). Tumor infiltrated lymphocytes (TILs) were isolated at 24 hours after the second dose and analyzed by flow cytometry. Compared with IgG control, both CD4+ and Cd8+ T cell subpopulation in tumor tissue increase in response to ATG-031, while the proportion of regulatory T cells was reduced by ATG-031. (B0D) Single cell RNA-seq result of TILs isolated from MC38-hCD24 tumor 4 hours or 24 hours post IgG or 3mg/kg ATG-031 treatment. (B0 2D-UMAP embedding plot of TILs color by cell types. T cell subpopulation is circled (C) Percentage of cell subpoplulation of TILs from each treatment group, quantified from 2D-UMAP. n=4 for ATG-031_24h and n=3 for other groups. (D) Cell-level Pathway activity of T cell population, inference by PROGENy. ATG-031 treatment upregulated Estrogen signaling and reduced PI3K signaling compared with IgG, suggesting an increase of T cell activation in response to ATG-031.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.