Quantitative Analysis of Binding Kinetics Behavior of CD47 by Atomic Force Microscopy

Immunotherapy is an effective approach for treating cancer. The interaction between the cluster of differentiation 47 and signal regulatory protein α constitutes a natural immune checkpoint, which plays a crucial role in the immune escape associated with tumors. In this study, we employed a lab-made atomic force microscope single-molecule force spectroscopy technique to investigate the interactions among CD47, anti-CD47 antibodies, and SIRPα in a liquid environment. Our results revealed that the CD47/anti-CD47 antibody complex exhibits a stronger affinity and greater binding stability compared with the CD47/SIRPα complex. We quantitatively measured the unbinding forces associated with the specific interactions of SIRPα and anti-CD47 antibodies with CD47 on the A549 cells. These force measurements on cells yielded results similar to those obtained on the substrate, further highlighting the difference in binding stability between the complexes. Concurrently, mechanical measurements indicated that CD47 molecules are relatively dispersed on the surface of the A549 cells. Following interferon-gamma treatment, the frequency of CD47 recognition events on A549 cells increased significantly, demonstrating that IFN-γ upregulated the CD47 expression on these cells. Collectively, these findings provide deeper insights into the molecular mechanism governing the interactions between CD47, its antibody, and SIRPα. This work establishes a foundation for the development of tumor immune checkpoint inhibitors targeting this pathway.