Site Specific Conjugation of Fluoroprobes to the Remodeled Fc N-Glycans of Monoclonal Antibodies Using Mutant Glycosyltransferases: Application for Cell Surface Antigen Detection

Abstract

The Fc N-glycan chains of four therapeutic monoclonal antibodies (mAbs), namely, Avastin, Rituxan, Remicade, and Herceptin, released by PNGase F, show by MALDI analysis that these biantennary N-glycans are a mixture of G0, G1, and G2 glycoforms. The G0 glycoform has no galactose on the terminal GlcNAc residues, and the G1 and G2 glycoforms have one or two terminal galactose residues, respectively, while no N-glycan with terminal sialic acid residue is observed. We show here that under native conditions we can convert the N-glycans of these mAbs to a homogeneous population of G0 glycoform using β1,4 galactosidase from Streptococcus pneumoniae. The G0 glycoforms of mAbs can be galactosylated with a modified galactose having a chemical handle at the C2 position, such as ketone or azide, using a mutant β1,4 galactosyltransferase (β1,4Gal-T1-Y289L). The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group. The linking of a biotinylated or a fluorescent dye carrying derivatives selectively occurs with the modified galactose, C2-keto-Gal, at the heavy chain of these mAbs, without altering their antigen binding activities, as shown by indirect Enzyme Linked Immunosorbent Assay (ELISA) and Fluorescence Activated Cell Sorting (FACS) methods. Our results demonstrate that the linking of cargo molecules to mAbs via glycans could prove to be an invaluable tool for potential drug targeting by immunotherapeutic methods.

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…They first homogenize mAb glycoforms via galactosidase digestion and subsequently integrate keto-or azide-modified galactose residues using mutant galactosyltransferases (40,41). Similarly, a recent report by Senter and co-workers exploits the promiscuity of native fucosyltransferases to incorporate thiol-functional fucose analogues for thiolmaleimide conjugation to MMAE (42).…”

Straightforward Glycoengineering Approach to Site-Specific Antibody–Pyrrolobenzodiazepine Conjugates

“…Subsequent conjugation of a doxorubicin payload using strain-promoted azide−alkyne cycloaddition (SPAAC) resulted in an ADC with a drug-toantibody ratio (DAR) of 4. 15 Recently, SynAffix reported a strategy in which the N-glycan was partially deglycosylated by endoglycosidase S (EndoS), and fluorinated GalNAz derivatives ( Figure 1) were installed enzymatically using a mutant β1,4-galactosyltransferase (GalT(Y289L)), 13,16 followed by conjugation of monomethyl auristatin F to the antibody using SPAAC, yielding ADCs with a DAR of 2. 17 Herein we describe a one-step chemoenzymatic method to generate ADCs with a drug-to-antibody ratio of 4 using an alkynyl pyrrolobenzodiazepine (PBD) dimer payload (SG3364, Scheme 1) conjugated to an azide-modified GalNAc (GalNAz, Figure 1) using copper-catalyzed click chemistry (Figure 2A,C).…”

Glycans of Antibodies as a Specific Site for Drug Conjugation Using Glycosyltransferases

“…32,33 The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group (Figure 4 A,B). The transferred C2-keto-Gal is coupled to any molecule carrying an aminooxy group, 29 e.g.…”

“…The transferred C2-keto-Gal is coupled to any molecule carrying an aminooxy group, 29 e.g. aminooxy-biotin, aminooxyfluoroprobes, [31][32][33][34] or aminooxy-drug 35, 36 ( Figure 4A). The GalNAz that was transferred from UDP-GalNAz to the GlcNAc residues on the mAb glycan with the mutant enzyme β4Gal-T1-Y289L is then conjugated with alkyne residue using Cu-catalyzed azidealkyne cycloaddition click chemistry 23 ( Figure 4B) or Cu-free strain-promoted azidealkyne Huisgen cycloaddition reaction.…”