Selection and Characterization of FD164, a High-Affinity Signal Regulatory Protein α Variant with Balanced Safety and Effectiveness, from a Targeted Epitope Mammalian Cell-Displayed Antibody Library

Abstract

Phagocytic resistance plays a key role in tumor-mediated immune escape, so phagocytosis immune checkpoints are a potential target for cancer immunotherapy. Cluster of differentiation 47 (CD47) is one of the important phagocytosis immune checkpoints, thus, blocking the interaction between CD47 and signal regulatory protein α (SIRPα) hopefully provides new options for cancer treatment. Using computer-aided targeted epitope mammalian cell-displayed antibody library, we screened and obtained an engineered SIRPα variant Fc fusion protein, FD164, with higher CD47-binding activity than wild-type SIRPα.Compared with wild-type SIRPα, FD164 has approximately 3-fold higher affinity for binding to CD47, which further enhanced its phagocytic effect in vitro and tumor suppressor activity in vivo. FD164 maintains the similar anti-tumor activity of the clinical research drug Hu5F9 in the mouse xenograft model. Furthermore, FD164 combined with rituximab can significantly improve the effect of single-agent therapy. On the other hand, compared with Hu5F9, FD164 does not cause hemagglutination, and its ability to bind to red blood cells or white blood cells is weaker at the same concentration. Finally, it was confirmed by computer structure prediction and alanine scanning experiments that the N 45 , E 47 , 52 TEVYVK 58 , K 60 , 115 EVTELTRE 122 , E 124 residues of CD47 are important for SIRPα or FD164 recognition. In a word, we obtained a high affinity SIRPα variant FD164 with balanced safety and effectiveness.