Methods for modifying human antibodies by glycan engineering

A method for glycan engineering the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at each Asn-297, wherein the oligosaccharide has the following structure:

• wherein Rf is selected from H or
• the method comprising:
• (a) reacting UDP galactose with the oligosaccharide having the structure:
• using β-1,4-galactosyltransferase as an enzymatic catalyst to form a Gal2-oligosaccharide having the structure:
• and
• (b) reacting the Gal2-oligosaccharide having the structure:
• with a compound having the structure:
• using an alpha-2,6-sialyltransferase as the enzymatic catalyst to form a glycan having the structure:
• wherein:
• R1 is selected from hydrogen or hydroxy;
• each instance of R2, R3, R4, and R5 is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, —N3, —NO2, —N(RB)2, —N(RA)C(O)RA, —ORA, —OC(O)RA, —SRA, —C(O)N(RB)2, —CN, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO2N(RB)2, and —NHSO2RB;
• RN is selected from —N3, —NO2, —N(RB)2, —N(RA)C(O)RA, —ORA, —OC(O)RA, —SRA, —C(O)N(RB)2, —CN, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO2N(RB)2, and —NHSO2RB;
• each instance of RA is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl;
• and
• each instance of RB is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl.

A method for engineering the glycan on the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at Asn-297, the method comprising:

• (a) providing a human, chimeric, or humanized glycosylated antibody;
• (b) contacting the antibody with a β-1,4-galactosyltransferase and UDP galactose thereby coupling the terminal unit of the oligosaccharide to the galactose to form an oligosaccharide having a terminal galactose unit, wherein the oligosaccharide having a terminal galactose unit is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody;
• and
• (c) contacting the antibody comprising the oligosaccharide having a terminal galactose with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose unit to form an oligosaccharide having a terminal Neu5Ac2Gal2, wherein the oligosaccharide having a terminal Neu5Ac2Gal2 is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody.