An anti-CD47 antibody binds to a distinct epitope in a novel metal ion-dependent manner to minimize cross-linking of red blood cells

Xiao Lu; Ziyue Chen; Chunyan Yi; Zhiyang Ling; Jing Ye; Kaijian Chen; Yao Cong; Sonam Wangmo; Shipeng Cheng; Ran Wang; Danyan Zhang; Jiefang Xu; Jichao Yang; Liyan Ma; Qing Duan; Xiaoyu Sun; Jianping Ding; Bing Sun

doi:10.1016/j.jbc.2025.110420

An anti-CD47 antibody binds to a distinct epitope in a novel metal ion-dependent manner to minimize cross-linking of red blood cells

J Biol Chem. 2025 Jun 25:110420. doi: 10.1016/j.jbc.2025.110420. Online ahead of print.

Authors

Xiao Lu¹, Ziyue Chen², Chunyan Yi¹, Zhiyang Ling¹, Jing Ye³, Kaijian Chen², Yao Cong², Sonam Wangmo³, Shipeng Cheng¹, Ran Wang⁴, Danyan Zhang³, Jiefang Xu⁴, Jichao Yang³, Liyan Ma¹, Qing Duan⁵, Xiaoyu Sun⁶, Jianping Ding⁷, Bing Sun⁸

Affiliations

¹ Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
² Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
³ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
⁴ Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
⁵ TOT BIOPHARM Company Limited, Jiangsu 215024, China.
⁶ Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China. Electronic address: sunxiaoyu@fudan.edu.cn.
⁷ Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address: jpding@sibcb.ac.cn.
⁸ Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address: bsun@sibs.ac.cn.

PMID: 40578556
DOI: 10.1016/j.jbc.2025.110420

Abstract

Cluster of differentiation 47 (CD47) is a widely expressed transmembrane protein that plays a crucial role in immune self-recognition. Cancer cells upregulate CD47 expression to promote immune escape through activating the "don't eat me" signal via interactions with signal regulatory protein α (SIRPα) on macrophages. The effectiveness of anti-CD47 antibodies has been demonstrated in multiple tumour models. However, since CD47 is also expressed in human red blood cells (RBCs) and platelets, the clinical application of anti-CD47 antibodies requires careful consideration of blood toxicity. One major obstacle to the clinical application of CD47 antibodies is the haemagglutination caused by RBCs cross-linking. In this study, we generated Hu1C8, a humanized anti-CD47 monoclonal antibody that demonstrated increased selectivity for binding to CD47 on cancer cells and lacked haemagglutination activity. Epitope mapping and the crystal structure of the Hu1C8 Fab-CD47 extracellular domain (ECD) complex revealed that Hu1C8 binds to a distinct epitope of CD47 in a Ca²⁺-dependent manner. The unique recognition and binding mode allowed Hu1C8 to bind CD47 on RBCs with reduced haemagglutination activity while still maintaining effective antitumour activity. These findings demonstrate a feasible strategy for developing CD47 antibodies with high antitumor activity but low RBC haemagglutination activity. Our study elucidates how epitope-specific antibody influences antibody-induced cell cross-linking, offering innovative strategies for antibody design to either leverage or avoid cell cross-linking effects.

Keywords: (6): CD47; bivalent metal ion; cell cross-linking; crystallography; non-RBC haemagglutination; rabbit monoclonal antibody.