Development of a High-Affinity Antibody-Binding Peptide for Site-Specific Modification

Area of Science:

• Bioconjugation Chemistry
• Protein Engineering
• Antibody Therapeutics

Background:

• Immunoglobulin G (IgG)-binding peptides facilitate antibody modification and homogeneous antibody-drug conjugate preparation.
• Existing peptides like 15-IgBP bind IgG via Lys8 but lack a primary amino group for crosslinking.
• Previous work identified 15-Lys8Leu with high affinity due to Leu8's γ-dimethyl group, but it also lost the essential primary amino group.

Purpose of the Study:

• To design and synthesize a novel peptide with both high IgG binding affinity and a primary amino group for crosslinking.
• To develop a versatile tool for site-specific antibody modification and the creation of homogeneous antibody-drug conjugates.

Main Methods:

• Design and synthesis of a novel unnatural amino acid, 4-(2-aminoethylcarbamoyl)leucine (Aecl).
• Synthesis of a peptide containing Aecl at position 8 (15-Lys8Aecl).
• Characterization of binding affinity and successful labeling of trastuzumab (Herceptin®) with FITC-labeled 15-Lys8Aecl(OSu).

Main Results:

• The novel peptide 15-Lys8Aecl demonstrated significantly higher binding affinity (K_d = 24.3 nM) compared to 15-IgBP (K_d = 267 nM).
• The synthesized peptide possesses both strong binding affinity and the necessary primary amino group for crosslinking.
• FITC-labeled 15-Lys8Aecl(OSu) successfully achieved site-specific labeling of the antibody trastuzumab.

Conclusions:

• The novel peptide scaffold, 15-Lys8Aecl, offers a promising solution for selective antibody modification.
• This peptide combines high affinity and crosslinking capabilities, making it valuable for developing homogeneous antibody-drug conjugates.
• The developed tool facilitates the site-specific conjugation of antibodies with various molecules of interest, including therapeutic drugs.