Glycosynthase variants for antibody-drug conjugate engineering

An engineered bioconjugate, comprising:

• a biomolecule and
• a modified glycan, coupled with the biomolecule;
• wherein the modified glycan comprises:
• (i) a first polyethylene glycol (PEG) moiety, and
• (ii) a first reactive moiety or a resultant moiety thereof from a biorthogonal reaction;
• wherein the resultant moiety comprises a triazole moiety, a DBCO-derived moiety, or a maleimide-derived moiety;
• wherein the biomolecule comprises an antibody or antigen binding fragment thereof, a protein, or a peptide;
• wherein the first reactive moiety comprises an azido group and is configured to interact with an alkyne moiety in a biorthogonal reaction;
• and
• wherein the biorthogonal reaction is a copper-free click chemistry.

A therapeutic conjugate comprising a formula of:

• wherein C is a reactive moiety, configured to react in a biorthogonal reaction;
• wherein L is a linker unit comprising a hydrophilic moiety, a cleavable moiety, and a spacer;
• wherein D is a therapeutic agent;
• and
• wherein the reactive moiety comprises an unsaturated moiety;
• optionally wherein the unsaturated moiety is an alkene moiety or an alkyne moiety;
• and/or wherein the reactive moiety is a non-native and nonperturbing chemical group;
• optionally wherein the reactive moiety is a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN), a cyclic alkyne, a maleimide group, a α,β-unsaturated carbonyl group, or a sulfonyl pyrimidine.