Introduction: The Evolution Toward Next-Generation ADCs

Antibody-drug conjugates (ADCs), often described as “biological missiles,” have become one of the most promising therapeutic platforms in oncology. By combining the targeting specificity of monoclonal antibodies with the potent cytotoxic activity of small-molecule drugs, ADCs enable selective drug delivery to tumor cells while minimizing systemic toxicity.

With the rapid expansion of the ADC pipeline, the demand for more precise, homogeneous, and controllable ADC conjugation technologies continues to grow. As of 2025, 19 ADC drugs have been approved worldwide, and more than 400 ADC candidates are currently under development, highlighting the increasing importance of advanced ADC manufacturing technologies.

However, traditional ADC conjugation approaches, including random lysine or cysteine conjugation, often suffer from several limitations:

• Batch-to-batch variability caused by heterogeneous conjugation sites
• Uncontrolled drug-to-antibody ratio (DAR)
• Reduced pharmacokinetic consistency
• Potential impact on antibody activity and safety profiles

Therefore, site-specific ADC conjugation has emerged as a key strategy for developing next-generation ADC therapeutics.

Recently, Endo S2 and microbial transglutaminase (mTGase) have attracted significant attention as powerful enzymatic tools for precision antibody modification. A breakthrough study published in ACS Chemical Biology (2026) demonstrated that Endo S2 and mTGase can function together through an orthogonal dual-enzyme conjugation strategy, enabling efficient construction of dual-payload ADCs.

Dual-Enzyme Orthogonal Conjugation Enables Dual-Payload ADC Construction

The innovative feature of this strategy is that Endo S2 and mTGase independently modify two neighboring sites within the antibody Fc region:

• mTGase-mediated conjugation at Q295
• Endo S2-mediated glycan remodeling and conjugation at N297

Although Q295 and N297 are spatially adjacent within the Fc region, the two enzymatic reactions remain highly compatible without mutual interference.

Using trastuzumab (Herceptin®) as a model antibody, researchers successfully generated a dual-payload ADC containing:

• MMAE, a microtubule inhibitor, conjugated through the Q295 site
• Exatecan, a topoisomerase I inhibitor, introduced through the N297 glycan site

This study provides a new enzymatic platform for designing ADCs with multiple payload combinations.

Key Advantages of Endo S2 and mTGase Dual-Enzyme ADC Conjugation

1. Highly Precise Site-Specific Modification

Compared with random conjugation methods, enzymatic ADC conjugation enables accurate control of modification sites.

Key findings include:

• mTGase achieved >99% Q295 conjugation selectivity
• Endo S2 transglycosylation efficiency remained unaffected after Q295 modification
• Antibody binding specificity was fully maintained after enzymatic modification

2. Homogeneous Dual-Payload ADC Generation

Both sequential and parallel reaction strategies successfully produced homogeneous dual-payload ADCs, demonstrating:

• Excellent enzyme compatibility
• Strong orthogonality
• Flexible manufacturing workflows

This approach enables researchers to develop ADC molecules with precisely controlled payload combinations and DAR values.

Why Dual-Payload ADCs Matter for Future Cancer Therapy

Tumor heterogeneity and drug resistance remain major challenges in cancer treatment.

Dual-payload ADC technology provides a potential solution by combining drugs with different mechanisms of action within a single antibody-based delivery system.

Potential advantages include:

• Enhanced tumor cell killing through complementary mechanisms
• Improved response against heterogeneous tumor populations
• Reduced likelihood of resistance development

The combination of Endo S2 and mTGase provides a modular enzymatic platform for next-generation ADC engineering.

Endo S2: A Precision Tool for Fc Glycan Remodeling

What is Endo S2?

Endo S2 is an IgG-specific endoglycosidase derived from Streptococcus pyogenes.

It specifically cleaves the Fc N-glycan structure between two internal N-acetylglucosamine (GlcNAc) residues while preserving the core Fucα1,6GlcNAc structure at the N297 site.

Unlike PNGase F, which completely removes Fc glycans, Endo S2 enables controlled glycan remodeling while maintaining:

• Native antibody structure
• Fc stability
• Biological functionality

Endo S2 for One-Step Glycan-Directed ADC Conjugation

One of the major advantages of Endo S2 is its ability to perform:

Fc glycan remodeling + site-specific conjugation in a single enzymatic process

By using functionalized glycan oxazoline substrates, Endo S2 can directly introduce payload-containing glycans onto the antibody Fc N297 site.

Benefits include:

• Reduced process complexity
• Fewer purification steps
• Improved ADC homogeneity
• Better control of DAR distribution

Endo S2 Applications

ADC Development

Generation of homogeneous site-specific ADCs through glycan-directed conjugation.

Antibody Characterization

Precise Fc glycan analysis using mass spectrometry-based workflows.

Antibody Engineering

Platform for Fc glycan remodeling and functional modification.

Diagnostic Reagent Development

Efficient labeling of antibodies with fluorescent dyes or biotin.

mTGase: A Precision “Molecular Glue” for Antibody Q295 Conjugation

What is mTGase?

Microbial transglutaminase (mTGase) is an enzyme that catalyzes the formation of stable isopeptide bonds between glutamine (Gln/Q) residues and primary amine-containing substrates.

For IgG antibodies, mTGase primarily recognizes the Fc Q295 site, enabling precise antibody conjugation without introducing engineered amino acid tags.

Advantages of mTGase-Based ADC Conjugation

Site-Specific Modification

mTGase enables selective Q295 modification with conjugation selectivity above 98%.

No Antibody Engineering Required

Natural antibodies can be directly modified without introducing non-natural amino acids or engineered tags.

Mild Reaction Conditions

Efficient conjugation under physiological conditions:

• PBS buffer
• pH 7.4
• 37°C

Controlled DAR Value

Reaction conditions can be optimized to achieve precise drug loading control.

Applications of mTGase

• Site-specific ADC development
• Protein labeling with biotin or fluorescent molecules
• Antibody-drug conjugation
• Dual-payload ADC construction with Endo S2

Hzymes Endo S2 Pro & mTGase: High-Performance Enzymes for Next-Generation ADC Development

To support advanced ADC research and manufacturing, Hzymes provides high-quality Endo S2 Pro and mTGase enzymes designed for precision antibody conjugation applications.

Hzymes Endo S2 Pro

Key Features

High enzymatic activity

• Strong hydrolysis and transglycosylation performance
• Compatible with diverse payload designs

Biopharmaceutical-grade quality

• Performance specifications meet GMP-oriented development requirements

Patent-friendly solution

• Proprietary engineered enzyme technology helps avoid wild-type enzyme patent limitations

Reliable supply capability

• Large-scale manufacturing capacity ensures stable supply for industrial applications

Hzymes mTGase

Key Features

High specific activity

• Meets diverse antibody conjugation requirements

Biopharmaceutical-grade quality

• Suitable for research, process development, and manufacturing applications

Industrial-scale production

• Ensures consistent quality and supply security

Conclusion: Enabling the Future of Precision ADC Development

The combination of Endo S2 and mTGase represents a powerful enzymatic strategy for next-generation ADC development.

By enabling precise, orthogonal, and modular antibody conjugation, this dual-enzyme platform provides new opportunities for:

• Homogeneous ADC manufacturing
• Dual-payload ADC design
• Improved therapeutic performance
• Scalable biopharmaceutical production

With advanced enzyme engineering capabilities, Hzymes is committed to providing reliable enzymatic solutions that accelerate innovation in ADC development and biopharmaceutical manufacturing.

Product Selection Guide

Reference

ACS Chemical Biology 2026, 21, 1474-1486. DOI:10.1021/acschembio.6c00229