Fc glycan remodeling platform method for site-specific antibody conjugation and applications thereof
AssigneeUniversity of Maryland, College Park
InventorsLai-Xi Wang, Xiao Zhang
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Abstract
The present disclosure provides a one-pot chemoenzymatic method for site-specific modification and conjugation of antibodies at their Fc glycan site to produce structurally well-defined antibody conjugates carrying defined drugs and other entities. The method is enabled by the discovery that certain endoglycosidases have the ability to both deglycosylate an antibody and to recognize selectively modified small disaccharide oxazolines for transglycosylation on antibodies without hydrolysis of the resulting products.
Claims10
A one pot Fc glycan remodeling method that uses a single enzyme for both deglycosylation and transglycosylation, to provide a remodeled site-specifically functionalized antibody, the method comprising the steps of:
- (a) providing a single reactor, container, column, or pot;
- (b) introducing a single endoglycosidase having both deglycosylation and transglycosylation activity;
- (c) introducing an antibody for deglycosylation by the single endoglycosidase, thereby providing a deglycosylated intermediate that contains at least one N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucα1,6GlcNAc) acceptor;
- (d) providing a modified glycan oxazoline comprising a disaccharide core;
- and
- (e) transglycosylating the modified glycan oxazoline to the N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucα1,6GlcNAc) acceptor by the introduced single endoglycosidase of step (b), to provide a remodeled site-specifically functionalized antibody;
- wherein:
- said modified glycan oxazoline is a Group II Glcβ1,4GlcNAc based disaccharide oxazolines, comprising a glucose-β1,4-N-acetylglucosamine disaccharide core and the enzyme is Endo S (SEQ ID NO. 4), or Endo S2 (SEQ ID NO. 1).
A one pot Fc glycan remodeling method that uses a single enzyme for both deglycosylation and transglycosylation to provide a remodeled site-specifically functionalized antibody, the method comprising the steps of:
- (a) providing a single reactor, container, column, or pot;
- (b) introducing a single endoglycosidase having both deglycosylation and transglycosylation activity;
- (c) introducing an antibody for deglycosylation by the single endoglycosidase, thereby providing a deglycosylated intermediate that contains at least one N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucα1,6GlcNAc) acceptor;
- (d) providing a modified glycan oxazoline comprising a disaccharide core;
- and
- (e) transglycosylating the modified glycan oxazoline to the N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucα1,6GlcNAc) acceptor by the introduced single endoglycosidase of step (b), to provide a remodeled site-specifically functionalized antibody;
- wherein said modified glycan oxazoline is a Group I Manβ1,4GlcNAc based disaccharide oxazolines comprising a mannose-β1,4-N-acetylglucosamine disaccharide core and the enzyme is Endo S2 (SEQ ID NO. 1).
A one pot Fc glycan remodeling method that uses a single enzyme for both deglycosylation and transglycosylation to provide a remodeled site-specifically functionalized antibody, the method comprising the steps of:
- (a) providing a single reactor, container, column, or pot;
- (b) introducing a single endoglycosidase having both deglycosylation and transglycosylation activity;
- (c) introducing an antibody for deglycosylation by the single endoglycosidase, thereby providing a deglycosylated intermediate that contains at least one N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucα1,6GlcNAc) acceptor;
- (d) providing a modified glycan oxazoline comprising a disaccharide core;
- and
- (e) transglycosylating the modified glycan oxazoline to the N-acetylglucosamine (GlcNAc) or core-fucosylated N-acetylglucosamine (Fucα1,6GlcNAc) acceptor by the introduced single endoglycosidase of step (b), to provide a remodeled site-specifically functionalized antibody;
- wherein said modified glycan oxazoline is a Group III Galβ1,4GlcNAc based disaccharide oxazolines, comprising a galactose-β1,4-N-acetylglucosamine disaccharide core wherein the enzyme is Endo S (SEQ ID NO. 4).
Dependencies are inferred from the claim text.