Cut Once, Double the Value: How Endo S and Endo S2 Leverage the Billion-Dollar ADC Market?

Endoglycosidases Endo S and Endo S2, derived from Streptococcus pyogenes, are highly efficient and specific tools for hydrolyzing the N-linked glycan at the Asn297 site of the IgG antibody Fc region.

These enzymes play multiple roles in the biopharmaceutical field: they serve as core tools for analyzing critical quality attributes of antibody drugs, act as "molecular scissors" in the production of next-generation site-specific ADCs (antibody-drug conjugates), and demonstrate clinical potential for treating autoimmune diseases.

The origin of Endo S and Endo S2 is rather dramatic. They were not designed by scientists for laboratory applications but evolved as immune evasion "weapons" of Streptococcus pyogenes. When this bacterium infects humans, it secretes Endo S and Endo S2 to precisely hydrolyze the N-glycan at the Asn297 site of the IgG antibody Fc region, preventing the antibody from binding to Fcγ receptors and thereby disabling its ability to recruit immune cells to clear the bacteria. Nature placed the functional switch of IgG antibodies on their glycans, and Streptococcus pyogenes found the "key."

In the development and quality control of antibody drugs, N-glycosylation modifications in the Fc region are one of the critical quality attributes affecting drug safety and efficacy. The level of core fucosylation directly determines the strength of antibody-dependent cell-mediated cytotoxicity (ADCC)—defucosylation significantly enhances ADCC, while high-mannose types accelerate drug clearance in vivo. Regulatory agencies require quantitative measurement of fucosylation and high-mannose levels in antibody drugs. This quality control demand has propelled Endo S and Endo S2 to the forefront of the industry.

In mass spectrometry workflows, Endo S/S2 is typically used in the first step of the "Middle-up" analysis strategy. After treating intact antibodies with Endo S/S2, only one GlcNAc residue (which may include fucose) remains on the Fc region, significantly simplifying the mass spectrum and enabling rapid identification of fucosylation and high-mannose levels. In more comprehensive antibody characterization protocols, Endo S2 is also used to prepare deglycosylated subunit samples, combined with IdeS proteolysis and reduction, enabling multi-level analysis from intact proteins to subunits and peptides.

A comparison between Endo S and PNGase F is noteworthy. PNGase F can cleave almost all N-linked glycans but has low digestion efficiency under native conditions; Endo S can completely deglycosylate murine monoclonal IgG under native conditions, whereas PNGase F cannot achieve complete digestion. Additionally, PNGase F cleaves the entire glycan from asparagine, releasing the glycan intact, while Endo S/S2 only cleaves the glycosidic bond within the chitobiose core, leaving a GlcNAc residue on the protein. This difference determines their applications: the former is suitable for comprehensive glycomics analysis, while the latter is ideal for mass spectrometry quantification and glycoengineering.

If Endo S/S2 are "scissors" in the field of analysis and characterization, in the ADC field, they have become "molecular artisans" with dual functions of "cutting and grafting."

Pathway 1: Wild-Type Endo S/S2 + Specific Glycan Oxazoline (Traditional "One-Pot" Strategy)

Studies have found that while wild-type Endo S efficiently hydrolyzes heterogeneous complex-type N-glycans on the IgG Fc, when high-mannose or hybrid-type glycan oxazolines are used as glycan donors, Endo S can directly perform transglycosylation with almost no hydrolysis of the product. Based on this unique substrate specificity difference, deglycosylation and transglycosylation can be completed sequentially in the same reaction system without isolating the deglycosylated intermediate or changing enzymes.

Similarly, wild-type Endo S2 has been reported to directly assemble the disaccharide LacNAc oxazoline onto antibodies, and Endo S2 loses its hydrolytic activity toward LacNAc-modified antibodies, enabling "one-step" glycoengineering of wild-type antibodies.

Pathway 2: Wild-Type Endo S2 + Disaccharide Substrate ("Single-Enzyme One-Step" ADC Construction)

In ADC development, this strategy has moved from the laboratory to industrial applications. The "single-enzyme one-step method": directly using wild-type Endo S2 to conjugate payload-linked disaccharide oxazoline substrates to antibodies in one step, greatly simplifying the process steps and eliminating the need to pre-introduce azide groups or perform subsequent click chemistry reactions.

Additionally, Qilu Pharmaceutical has reported a "one-enzyme one-step" method for synthesizing ADCs—directly incubating glycosyltransferase-active endoglycosidases with disaccharide-linker-drug to obtain the target ADC in one step, applicable to various toxins such as MMAE and EXD. Similarly, the Shanghai Institute of Materia Medica reported a "one-step" homogeneous ADC preparation method based on the LacNAc-VC-PAB-MMAE complex, where MMAE is directly connected to the antibody glycosylation site under Endo-S2 catalysis.

From immune evasion weapons of Streptococcus pyogenes to core tools for antibody drug analysis and characterization, and then to the "magic scissors" of next-generation site-specific ADC production—Endo S and Endo S2 have completed a remarkable transformation from natural evolutionary tools to human biotechnological tools. Endo S, with its high specificity for complex-type glycans, has become the "gold standard" for fucosylation quantification analysis; Endo S2, with its broader substrate adaptability, demonstrates irreplaceable value in comprehensive glycomics analysis and ADC glycoengineering. As companies like Innovent Biologics deepen their investments in glycan-specific conjugation technologies and glycoenzyme mutant technologies continue to evolve, the application boundaries of Endo S/S2 are still expanding. For researchers engaged in antibody drug development and bioanalysis, mastering the properties and selection strategies of these two enzymes will undoubtedly add a set of versatile tools to experimental design and process development.

Related link: https://mp.weixin.qq.com/s/Fx--xmauYrk-fRFsA7i7JA