One-Pot Conversion of Free Sialoglycans to Functionalized Glycan Oxazolines and Efficient Synthesis of Homogeneous Antibody–Drug Conjugates through Site-Specific Chemoenzymatic Glycan Remodeling

Abstract

Antibody−drug conjugates (ADCs) are an important class of therapeutic agents that harness the highly specific antigen targeting property of antibodies to deliver toxic drugs for targeted cell killing. Site-specific conjugation methods are highly desirable for constructing homogeneous ADCs that possess a welldefined antibody-to-drug ratio, stability, ideal pharmacological profile, and optimal therapeutic index. We report here a facile synthesis of functionalized glycan oxazolines from free sialoglycans that are key donor substrates for enzymatic Fc glycan remodeling and the application of an efficient endoglycosidase mutant (Endo-S2 D184M) for site-specific glycan transfer to construct homogeneous ADCs. We found that by a sequential use of two coupling reagents under optimized conditions, free sialoglycans could be efficiently converted to selectively functionalized glycan oxazolines carrying azide-, cyclopropene-, and norbornene-tags, respectively, in excellent yield and in a simple one-pot manner. We further demonstrated that the recently reported Endo-S2 D184 M mutant was highly efficient for Fc glycan remodeling with the selectively modified glycan oxazolines to introduce tags into an antibody, which required a significantly smaller amount of glycan oxazolines and a much shorter reaction time than that of the Endo-S D233Q-catalyzed reaction, thus minimizing the side reactions. Finally homogeneous ADCs were constructed with three different click reactions. The resulting ADCs showed excellent serum stability, and in vitro cytotoxicity assays indicated that all the three ADCs generated from the distinct click reactions possessed potent and comparable cytotoxicity for targeted cancer cell killing.

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

“…When LC−MS analysis indicated the completion of the transglycosylation with conversion yield >95%, the product was purified Synthesis of 43. To a solution of 34 (200 μg) in a mixture of 50 mM PB/DMSO (70 μL/25 μL) was added the drug payload (DBCO-PEG5-VC-PAB-MMAE) 43 (5.0 mg/mL, 4.6 μL, 10 equiv), and the reaction was incubated at room temperature. After LC−MS analysis indicated the completion of the reaction, the mixture was diluted with 50 mM PB (3 mL) and filtered using a 0.22 μm filter to remove most of the hydrophobic payload, and the residue was purified using protein A chromatography to give 43 (150 μg as measured using Nanodrop).…”

“…However, this method requires trimming of the N-glycans to the terminal GlcNAc-glycan forms, and an excess of modified sugar nucleotide and enzymes and long incubation time are usually needed to drive the reaction, which often leads to incomplete reaction and thus heterogeneity of the products. On the other hand, the endoglycosidase-catalyzed glycan remodeling strategy, , particularly the discovery of the Endo-S and Endo-S2 glycosynthase mutants, − has gained attention in recent years for generating various homogeneous antibody glycoforms for functional studies, including antibody labeling and the development of ADCs. ,,− Despite these successful applications, however, most of the current studies rely on the extraction of natural N-glycans, and for ADC preparation, the N-glycans have to be functionalized for drug conjugation. Thus, the exploitation of simpler donor substrates with a tailored number of tags is greatly demanded for site-specific antibody labeling and conjugation.…”

Egg yolk sialylglycopeptide: purification, isolation and characterization of N-glycans from minor glycopeptide species

“…[15][16][17][18] However, SGP might not be the only relevant glycopeptide target that can be extracted from egg yolks. Early reports of the isolation of SGP indicated the presence of other glycopeptides bearing monosialylated glycans, 19,20 some of which allegedly corresponded exclusively to the antennary extension of the α-1,3 mannose arm of the N-glycan core SGP-A2 [3]G1S1 (Fig. 1).…”

“…Synthetically, SGP offers convenient access to N-glycan oxazolines, needed for glycoprotein remodelling by ENGase mediated transglycosylation. 2,3 Furthermore, SGP has been used as a precursor for the synthesis of constructs such as sialyl Lewis-X structures (S-Lex), 4 antennary polylactosamine repeats 5 and triantennary glycans, 6,7 or a combination of all the above, 8 providing access to N-glycan structures for which there is no natural source. Analytically, it has been used for testing new matrices for MALDI-MS, 9 for sialic acid derivatization strategies [10][11][12] and as a control for N-glycan release and labelling by reductive amination.…”

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

“…The N-methyl spacer (5) was specifically chosen to provide linker stability without premature release. [11] For the synthesis of the tetravalent rhamnose cluster ( 13), the tri-lysine core (7) was reacted with NHS-activated rhamnose derivative (10) to give the rhamnose cluster (11). After de-O-acetylation followed by removal of the Fmoc group, the resulting compound (12) was reacted with the NHS activated ester (2) to give the DBCO-tagged rhamnose cluster (13) after LH20 size exclusion chromatography (Scheme 1c).…”