Rational Redesign of an Fc-Binding Peptide for Multivalent Antibody Assembly

Abstract

Multivalent antibody assemblies offer opportunities to enhance avidity, organize immune complexes, and modulate higher-order protein interactions, but constructing such architectures from existing immunoglobulin G (IgG) molecules without redesigning the antibody scaffold remains challenging. Here, we report the rational redesign of a Protein A-derived Fc-binding peptide into ADP1, a stable dimeric Fc-binding peptide that directs Fc-mediated antibody assembly. ADP1 was designed from the parent Fc-binding peptide Z34C by preserving the Fc-recognition surface while redesigning the opposite helical surface to promote peptide-peptide association. Biophysical characterization showed that ADP1 retained nanomolar Fc-binding affinity while exhibiting markedly enhanced chemical and proteolytic stability compared with the parent peptide. Structural analyses of ADP1-Fc complexes revealed that ADP1 bridges neighboring Fc regions through a combined ADP1-Fc and ADP1-ADP1 interface, generating spiral higher-order Fc assemblies. This assembly principle was further extended to full-length IgG, where ADP1 promoted higher-order antibody association in a concentration-sensitive manner. In addition, covalent ADP1 functionalization enabled Fc-directed modification of full-length IgG while retaining Fab-mediated antigen recognition, demonstrating the utility of ADP1 as an antibody assembly and functionalization module. Finally, competitive addition of the parent Z34C peptide modulated ADP1-driven antibody assembly, suggesting a potential route for tuning Fc-mediated assembly propagation. Together, this work establishes a redesigned Fc-binding peptide platform for directing multivalent antibody assembly and functionalization without genetic reengineering of the IgG scaffold. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/737354v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@57cadeorg.highwire.dtl.DTLVardef@1c7d8e6org.highwire.dtl.DTLVardef@170fb85org.highwire.dtl.DTLVardef@90e6d6_HPS_FORMAT_FIGEXP M_FIG C_FIG