Site-Specific Chemical Conjugation of Antibodies by Using Affinity Peptide for the Development of Therapeutic Antibody Format

Abstract

Artificially modified IgG molecules are increasingly utilized in industrial and clinical applications. In the present study, the method of chemical conjugation by affinity peptide (CCAP) for site-specific chemical modification has been developed by using a peptide that bound with high affinity to human IgG-Fc. This method enabled a rapid modification of a specific residue (Lys248 on Fc) in a one-step reaction under mild condition to form a stable amide bond between the peptide and Fc. The monovalent peptide-IgG conjugate not only maintained complete antigen binding but also bound to Fc receptors (FcRn, FcγRI, and FcγRIIIa), indicating that it is a suitable conjugate form that can be further developed into highly functional antibody therapeutics. CCAP was applied for the preparation of an antibody-drug conjugate and a bispecific antibody to demonstrate the usefulness of this method.

Recent Chemical Approaches for Site‐Specific Conjugation of Native Antibodies: Technologies toward Next‐Generation Antibody–Drug Conjugates

“…To avoid the use of UV irradiation, Yu et al . and Kishimoto et al . reported the use of a combination of an affinity peptide with the activated ester method to modify a lysine residue in a mAb (Figure B).…”

Peptide‐Directed DNA‐Templated Protein Labelling for The Assembly of a Pseudo‐IgM

“…Previously, it has been employed as an affinity ligand in column purification of IgGs and as a half‐life‐increasing tag when expressed in fusion with a protein of interest (POI) . Very recently, methods to covalently link Fc binding peptides to the IgG through either a photo‐crosslinker or NHS‐coupling were demonstrated . In both approaches the directing FC‐III peptide is covalently linked to the antibody.…”

“…The method relies on the templated reaction of DNA guided by protein‐binding peptides. Contrary to the previously reported peptide‐directed conjugation to antibodies where the peptide is covalently attached to the antibody, this method only uses the peptide to direct the conjugation reaction. The method was found to be both robust and applicable across a selection of proteins, including humanized antibodies of the IgG1 and IgG2 isotypes as well as murine IgG1 and recombinant human albumin.…”

Peptide‐Directed DNA‐Templated Protein Labelling for The Assembly of a Pseudo‐IgM

“…[17] Very recently, methods to covalently link Fc binding peptides to the IgG through either ap hoto-crosslinker [18] or NHS-coupling were demonstrated. [19] In both approaches the directing FC-III peptide is covalently linked to the antibody.Very recently,the peptide was applied to conjugate small-molecule drugs to 1) Under dilute conditions, ap rotein of interest (POI, purple mAb) is targeted with an ODN bearing aprotein-bindingpeptide functioning as aguiding moiety (FC-III K, the blue b-sheet structure also illustrated in the inset, and its guiding strand). 2) Subsequently,areactive strand (red), functionalized with an aldehyde, is annealed to the guiding strand.…”

“…In summary,w eh ave developed an ew method for the conjugation of DNAtovarious proteins.The method relies on the templated reaction of DNAg uided by protein-binding peptides.C ontrary to the previously reported peptidedirected conjugation to antibodies where the peptide is covalently attached to the antibody, [18,19] this method only uses the peptide to direct the conjugation reaction. The method was found to be both robust and applicable across aselection of proteins,including humanized antibodies of the IgG1 and IgG2 isotypes as well as murine IgG1 and recombinant human albumin.…”