One-pot N-glycosylation remodeling of IgG with non-natural sialylglycopeptides enables glycosite-specific and dual-payload antibody–drug conjugates

Abstract

Chemoenzymatic transglycosylation catalyzed by endo-S mutants is a powerful tool for in vitro glycoengineering of therapeutic antibodies. In this paper, we report a one-pot chemoenzymatic synthesis of glycoengineered Herceptin using an egg-yolk sialylglycopeptide (SGP) substrate. Combining this one-pot strategy with novel non-natural SGP derivatives carrying azido or alkyne tags, glycosite-specific conjugation was enabled for the development of new antibody-drug conjugates (ADCs). The site-specific ADCs and semi-site-specific dual-drug ADCs were successfully achieved and characterized with SDS-PAGE, intact antibody or ADC mass spectrometry analysis, and PNGase-F digestion analysis. Cancer cell cytotoxicity assay revealed that small-molecule drug release of these ADCs relied on the cleavable Val-Cit linker fragment embedded in the structure. These results represent a new approach for glycosite-specific and dual-drug ADC design and rapid synthesis, and also provide the structural requirement for their biologic activities.

Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody–drug conjugates

“…As shown in the SDS-PAGE gel of Figure 8c, the heavy-chain band of PNGase-F-treated 5a completely shifted to a lower position, suggesting the complete release of N -glycan without any nonenzymatic glycation being observed. Furthermore, LC–MS analysis of the resulting Herceptin-Asp297 (see literature data 26 ) and azido-glycan (Fig. 8d) clearly verifies the enzymatic product that links the N -glycan to the GlcNAc motif of 2a .…”

“…Next, endoglycosidase mutants 28 , also called glycosynthases—which lack hydrolytic activity but possess trans-glycosylation activity through the use of a homogeneous synthetic N -glycan oxazoline as the activated substrate—are used to assemble this intact N -glycan substrate on the GlcNAc-bearing glycoprotein generated by the aforementioned deglycosylation by the WT enzyme to accomplish the glycoengineering. Endo-β- N -acetylglucosaminidase from Streptococcus pyogenes (Endo-S), as one of these endoglycosidases, specifically recognizes the IgG Fc domain and has been used for efficient glyco-remodeling of therapeutic antibodies 21–26 . The glycan substrate specificity of Endo-S covers a wide range of natural and modified N -glycan structures.…”

“…Recently, we reported a one-pot chemoenzymatic method using azido-tagged N -glycopeptide precursors, and successive ‘click’ conjugation with alkyne-tagged small molecules for the synthesis of gsADCs 26 . The gsADC concept was originally developed using glycosyltransferase to introduce azido-tagged monosaccharides on external residues of IgG N -glycans, followed by small-molecule drug conjugation 30–32 .…”

“…Since the original method was published, we and other scientists have made substantial improvements, including the development of one-pot chemoenzymatic procedures 26 , use of new endoglycosidases 25 and improved fucosidases 23,25 , identification of various N -glycan substrates 23,26 , the introduction of different types of modification chemistry 24,37,38 , and an optimized procedure for N -glycan extraction 37 , to broaden the adaptation of this method. We report here the updated protocol describing all required steps of this approach.…”

“…Preparation of GlcNAc-IgG from deglycosylation of native IgG by WT Endo-S and α-L-fucosidase AlfC is described in Steps 46–62. The cloning, expression, and purification of the enzymes Endo-M, α-l-fucosidase AlfC, Endo-S WT, and its mutant D233Q are performed as described in the literature 25,26,28 with modification as described in the Supplementary Results. Enzyme activity is critical to the deglycosylation and transglycosylation steps.…”