General and Robust Chemoenzymatic Method for Glycan-Mediated Site-Specific Labeling and Conjugation of Antibodies: Facile Synthesis of Homogeneous Antibody–Drug Conjugates

Abstract

Site-specific labeling and conjugation of antibodies are highly desirable for fundamental research and for developing more efficient diagnostic and therapeutic methods. We report here a general and robust chemoenzymatic method that permits a one-pot site-specific functionalization of antibodies. A series of selectively modified disaccharide oxazoline derivatives were designed, synthesized, and evaluated as donor substrates of different endoglycosidases for antibody Fc glycan remodeling. We found that among several endoglycosidases tested, wild-type endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) exhibited remarkable activity in transferring the functionalized disaccharides carrying site-selectively modified azide, biotin, or fluorescent tags to antibodies without hydrolyzing the resulting transglycosylation products. This discovery, together with the excellent Fc deglycosylation activity of Endo-S2 on recombinant antibodies, allowed direct labeling and functionalization of antibodies in a one-pot manner without the need of intermediate and enzyme separation. The site-specific introduction of varied numbers of azide groups enabled a highly efficient synthesis of homogeneous antibody–drug conjugates (ADCs) with a precise control of the drug-to-antibody ratio (DAR) ranging from 2 to 12 via a copper-free strain-promoted click reaction. Cell viability assays showed that ADCs with higher DARs were more potent in killing antigen-overexpressed cells than the ADCs with lower DARs. This new method is expected to find applications not only for antibody–drug conjugation but also for cell labeling, imaging, and diagnosis.

Synthesis and Evaluation of Three Azide-Modified Disaccharide Oxazolines as Enzyme Substrates for Single-Step Fc Glycan-Mediated Antibody-Drug Conjugation

“…Our recent study has shown that wild-type Endo-S2 could accommodate selectively modified disaccharide oxazolines corresponding to the natural disaccharide (Manβ1,4GlcNAc) core for transglycosylation without product hydrolysis. 18 However, whether this enzyme could recognize and transfer unnatural core disaccharide structures to antibodies remains to be tested. We sought to test simpler disaccharide derivatives, such as Glcβ1,4GlcNAc and Galβ1,4GlcNAc (LacNAc) oxazolines, which are much easier to synthesize than the Manβ1,4GlcNAc core.…”

“…18 The resulting azide-tagged antibodies can be efficiently converted to homogeneous ADCs with different antibody/ drug ratios by subsequent click reactions. 18 To further examine the scope of this method, we report herein the synthesis and evaluation of selectively modified new disaccharide oxazolines, including the Glc-β1,4-GlcNAc and Gal-β1,4-GlcNAc (Lac-NAc) disaccharides, as substrates for enzymatic Fc-glycan remodeling of antibodies. We found that wild-type Endo-S2 had a remarkable flexibility to accommodate the "unnatural core disaccharides" for transglycosylation to provide azidefunctionalized antibodies (Figure 1).…”

Synthetic Antibody‐Rhamnose Cluster Conjugates Show Potent Complement‐Dependent Cell Killing by Recruiting Natural Antibodies

“…The use of the selectively azide‐tagged bi‐antennary N‐glycan for Fc glycan remodeling and subsequent conjugation has several advantages, including the site‐specific conjugation and the preservation of the natural Fc N‐glycan core after remodeling, which could be important for maintaining antibody stability and favorable pharmacokinetic property. Our previous studies have demonstrated that glycosynthase EndoS2‐D184 M possesses quite relaxed substrate specificity in transferring different natural and selectively modified glycan oxazolines, [10,14] Recently, we have shown that both the wild type and the EndoS2‐D184 M mutant of Endo‐S2 can efficiently transfer selectively azide‐functionalized Manβ1,4‐GlcNAc oxazoline to the deglycosylated antibody for constructing homogeneous antibody‐drug conjugates [14a] . This method could provide an alternative approach for making the antibody‐rhamnose or αGal cluster conjugates.…”

Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases

“…Accordingly, IgG-specific ENGases have been shown to ameliorate autoimmune disease in diverse animal models [10][11][12][13] . In addition, EndoS and EndoS2 are powerful tools for the chemoenzymatic synthesis of antibodies with homogenous glycoforms [14][15][16][17] and drug conjugates 18,19 . The Fc region N-glycans of IgG antibodies in serum are composed of more than 33 distinct glycoforms 20 and their unique chemical structures modulate the effector functions by altering Fc binding to FcgRs, as well as the stability and half-life of the antibody 21 .…”