Chemoenzymatic glycoengineering of antibodies and FC fragments thereof
The present invention provides for recombinant Endo-S mutants that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sialylated oxazoline or synthetic oligosaccharide oxazoline is added to a core fucosylated or nonfucosylated GlcNAc-protein acceptor. Such recombinant Endo-S mutants are useful for efficient glycosylation remodeling of IgG1-Fc domain to provide different antibody glycoforms carrying structurally well-defined Fc N-glycans.
1. An intravenous immunoglobulin (IVIG)-containing composition, said composition comprising homogeneous Fc-sialylated glycoforms, a Streptococcus pyogenes Endoglycosidase-S (Endo-S) Asp-233 mutant, a deglycosylated IVIG having a GlcNAc-moiety positioned on the Fc region, and a sialoglycan oxazoline having a predetermined number of sugar residues, wherein the Streptococcus pyogenes Endo-S mutant is selected from a mutant comprising a D233Q (SEQ ID NO:2) or D233A (SEQ ID NO: 3) site-directed mutation.
2. The IVIG-containing composition of claim 1 , wherein the sialylated Fc glycoforms are at least 80% homogeneous.
3. The IVIG-containing composition of claim 1 , wherein the sialylated Fc glycoforms are at least 85% homogeneous.
4. The IVIG-containing composition of claim 1 , wherein the sialylated Fc glycoforms are at least 90% homogeneous.
5. The IVIG-containing composition of claim 1 , wherein the sialylated Fc glycoforms are at least 95% homogeneous.
6. The IVIG-containing composition of claim 1 , wherein the GlcNAc-moiety is either fucosylated or nonfucosylated.
7. The IVIG-containing composition of claim 1 , wherein the homogeneous Fc-sialylated glycoforms comprise sialylated-IVIG.
8. The IVIG-containing composition of claim 7 , wherein the sialylated-IVIG further comprises an additional biologically active agent or tag.
9. The IVIG-containing composition of claim 8 , wherein the additionally biologically active agent or tag is a drug, toxin, fluorescent probe, biotin, PEG, lipid or polypeptide.
10. The IVIG-containing composition of claim 1 , wherein the GlcNAc-moiety is fucosylated and is an alpha-1-6-fucosyl-GlcNAc-protein.
11. The IVIG-containing composition of claim 1 , wherein the IVIG further comprises an additional moiety selected from a group consisting of a therapeutic agent for treating cancer, a therapeutic agent for HIV, a toxin, an antibody different from the modified antibody which is reactive to another receptor, an antigen, a chemokine and a cytokine.