Journal of Biological Chemistry

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 tumor 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 hemagglutination 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 hemagglutination 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 hemagglutination activity while still maintaining effective antitumor activity. These findings demonstrate a feasible strategy for developing CD47 antibodies with high antitumor activity but low RBC hemagglutination 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.

bivalent metal ion

CD47

cell cross-linking

crystallography

non-RBC hemagglutination

rabbit monoclonal antibody

Antibody-dependent cellular phagocytosis

Cluster of differentiation 47

complementarity-determining region

Flow cytometry sorting

integrin-associated protein

peripheral blood mononuclear cells

polymerase chain reaction

© 2025 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biologyé