Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection.
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 beta1,4 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 beta1,4-galactosyltransferase (beta1,4Gal-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.
Structural Glycobiology Section, CCR-Nanobiology Program, SAIC-Frederick, Inc., Frederick, Maryland 21702, USA.
Elizabeth
E
Boeggeman
Boopathy
B
Ramakrishnan
Marta
M
Pasek
Maria
M
Manzoni
Anu
A
Puri
Kristin H
KH
Loomis
Timothy J
TJ
Waybright
Pradman K
PK
Qasba
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D000954:Antigens, Surface / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D019921:Biotinylation; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D013379:Substrate Specificity; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D005456:Fluorescent Dyes; D005690:Galactose; D005944:Glucosamine; D016695:Glycosyltransferases; D007074:Immunoglobulin G; D050505:Mutant Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D018719:Erb-b2 Receptor Tyrosine Kinases; D042461:Vascular Endothelial Growth Factor A