One-step synthesis of site-specific antibody–drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates

Shi, Wei; Li, Wanzhen; Zhang, Jianxin; Li, Tiehai; Song, Yakai; Zeng, Yicheng; Dong, Qian; Zeng, Lin; Gong, Likun; Fan, Shuquan; Tang, Feng; Huang, Wei

doi:10.1016/j.apsb.2021.12.013

Cited by 29 publications

(28 citation statements)

References 35 publications

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“…The filtrated was concentrated, and the residue was purified by flash column chromatography on silica gel (hexanes/EtOAc = 15:1−8:1) to give 18 (328 mg, 68% for 3 steps) as a colorless syrup. R f = 0.40 (hexanes/ EtOAc = 4:1); 1 (19). A solution of 18 (300 mg, 0.262 mmol) in a mixture of AcSH/pyridine/CHCl 3 (0.8 mL/ 0.6 mL/0.8 mL) was stirred at 50 °C for 14 h. After the completion of the reaction as monitored by TLC, the resulting mixture was concentrated and the residue was subjected to flash chromatography on silica gel (hexanes/EtOAc = 4:1− 1:1) to afford 19 (240 mg, 79%) as a colorless syrup.…”

Section: -O-2-[2-(2-azidoethoxy)ethoxy]ethyl-β-d-glucopyranosyl-(1 → ...mentioning

confidence: 99%

“…During the preparation of this manuscript, Shi and co-workers have reported a nice and independent work on Endo-S2 catalyzed transfer of drug-modified Gal-β1,4-GlcNAc oxazolines to antibody to form antibody-drug conjugates, in which the drug and antibody are conjugated through an oxime linkage. 19…”

Section: ■ Introductionmentioning

confidence: 99%

“…This method opens a new avenue to producing homogeneous antibody-drug conjugates, which represents a truly single-step and site-specific bioconjugation of a payload to intact antibodies. During the preparation of this manuscript, Shi and co-workers have reported a nice and independent work on Endo-S2 catalyzed transfer of drug-modified Gal-β1,4-GlcNAc oxazolines to antibody to form antibody-drug conjugates, in which the drug and antibody are conjugated through an oxime linkage …”

Section: Introductionmentioning

confidence: 99%

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

Zhang

Liu

et al. 2022

Bioconjugate Chem.

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Antibody-drug conjugates (ADCs) hold great promise for targeted cancer cell killing. Site-specific antibody-drug conjugation is highly desirable for synthesizing homogeneous ADCs with optimal safety profiles and high efficacy. We have recently reported that azide-functionalized disaccharide oxazolines of the Manβ1,4GlcNAc core were an efficient substrate of wild-type endoglycosidase Endo-S2 for Fc glycan remodeling and conjugation. In this paper, we report the synthesis and evaluation of new disaccharide oxazolines as enzyme substrates for examining the scope of the site-specific conjugation. Thus, azide-functionalized disaccharide oxazolines derived from Manβ1,4GlcNAc, Glcβ1,4GlcNAc, and Galβ1,4GlcNAc (LacNAc) were synthesized. Enzymatic evaluation revealed that wild-type Endo-S2 demonstrated highly relaxed substrate specificity and could accommodate all the three types of disaccharide derivatives for transglycosylation to provide site-specific azide-tagged antibodies, which were readily clicked with a payload to generate homogeneous ADCs. Moreover, we also found that Endo-S2 was able to accommodate drug-preloaded minimal disaccharide oxazolines as donor substrates for efficient glycan transfer, enabling a single-step and site-specific antibody-drug conjugation without the need of an antibody click reaction. The ability of Endo-S2 to accommodate simpler and more easily synthesized disaccharide oxazoline derivatives for Fc glycan remodeling further expanded the scope of this bioconjugation method for constructing homogeneous antibody-drug conjugates in a single-step manner. Finally, cell-based assays indicated that the synthetic homogeneous ADCs demonstrated potent targeted cancer cell killing.

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Section: -O-2-[2-(2-azidoethoxy)ethoxy]ethyl-β-d-glucopyranosyl-(1 → ...mentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Zhang

Liu

et al. 2022

Bioconjugate Chem.

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“…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 .…”

Section: Introductionmentioning

confidence: 99%

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

Trastoy

Cifuente

et al. 2022

Preprint

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Bacterial pathogens have evolved diverse and intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes secretes two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-linked glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analysis, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.

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“…The payload installation via these platforms usually follows a two-step procedure, in which bioorthogonal reaction handles, e.g., azide, were first introduced onto Fc via unnatural glycosylation, and drugs were further installed by bioorthogonal reactions, except the recently reported one-step EndoGS-based methods [19][20][21] . However, most of these platforms take hours to days to transfer smallmolecule payloads (<2000 Da), and are limited to direct transfer of unnatural donor substrates bearing biomacromolecules (>10,000 Da) in one-step.…”

mentioning

confidence: 99%

An Unexpected Single-step Glycosylation Enables the Construction of Antibody-Biomacromolecule Conjugates as Therapeutics

Yang

Song²,

Tian

et al. 2022

Preprint

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Glycoengineering has been demonstrated as a powerful approach to construct site-specific antibody conjugates. However, most glycoengineering strategies take of hours to days to achieve full conversion. Here, using unnatural fucosylation as an example, we show that reduce the complexity of Fc glycan dramatically boost its enzymatic conjugation. The fucosyl-biotinylation reaction on the created simple monoantennary LacNAc could be finished in 5 min while it takes 16 h on the natural biantennary LacNAc. More importantly, this unnatural fucosylation of the created simple LacNAc on antibody accommodate payloads over 15,000 Da, which allows the enzymatic construction of antibody-oligonucleotide conjugate (AOC) and bispecific antibody. Further applications of antibody conjugates in animal model and human cancer organoid indicates the translational potential of this platform, especially for constructing unlimited antibody-biomacromolecules (X) conjugate, "AXC", as therapeutics.

show abstract

One-step synthesis of site-specific antibody–drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates

Cited by 29 publications

References 35 publications

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

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

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

An Unexpected Single-step Glycosylation Enables the Construction of Antibody-Biomacromolecule Conjugates as Therapeutics

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