Enzymatic Construction of Dual-Payload Antibody-Drug Conjugates Using Endo S2 and Microbial Transglutaminase

A dual-payload antibody-drug conjugate is an engineered bioconjugate composed of:

- A monoclonal antibody that recognizes a tumor-associated antigen.

- Two chemically or pharmacologically distinct payloads.

- Linkers connecting each payload to defined antibody positions.

- A conjugation strategy that controls payload number and attachment site.

Unlike conventional ADCs carrying one type of cytotoxic molecule, dual-payload ADCs deliver two different therapeutic agents through the same antibody-targeting system.

The payloads may act through complementary mechanisms, such as microtubule disruption and DNA damage, or combine direct cytotoxic activity with an immune-stimulating function. The objective is not simply to increase the total amount of drug attached to the antibody, but to create a coordinated therapeutic system with a controlled molecular architecture.

Preclinical studies have shown that homogeneous dual-payload ADCs can outperform individual single-payload ADCs or mixtures of two separate ADCs in models involving heterogeneous antigen expression and drug-resistant tumor cells.

Reference : ScienceDirecte

Tumor antigen heterogeneity

Tumors are not uniform populations of identical cells. Antigen expression can vary between patients, between different lesions in the same patient and even among cells within a single tumor.

An ADC that is highly active against cells with strong antigen expression may be less effective against neighboring cells expressing lower levels of the target. This variation can leave surviving cellular populations capable of driving tumor recurrence.

Dual-payload ADCs can be designed so that one payload produces a membrane-permeable bystander effect, while the other remains active in resistant or strongly antigen-positive cells. This complementary activity may broaden the range of tumor cells affected by a single ADC construct.

Reference : Nature

Acquired drug resistance

Cancer cells can become resistant to ADC treatment through several mechanisms, including:

- Reduced target-antigen expression

- Impaired ADC internalization

- Altered lysosomal processing

- Increased drug-efflux transporter activity

- Changes in the molecular target of the payload

- Enhanced DNA repair or anti-apoptotic signaling

Using two payloads with different mechanisms of action may reduce dependence on a single cytotoxic pathway. However, dual-payload design does not automatically eliminate resistance. Payload selection, linker cleavage, intracellular release and the biological characteristics of the target tumor must all be evaluated experimentally.

Traditional ADC conjugation commonly targets naturally occurring lysine residues or reduced interchain cysteines. These stochastic reactions may generate complex mixtures containing molecules with different conjugation sites and different drug-to-antibody ratios.

Such heterogeneity can complicate:

- Analytical characterization

- Batch-to-batch consistency

- Pharmacokinetic interpretation

- Stability studies

- Toxicity assessment

- Process control during manufacturing

Site-specific enzymatic conjugation limits modification to defined antibody positions. Microbial transglutaminase has consequently become an important tool for producing homogeneous and structurally defined antibody conjugates.

The source document underlying this article presents high-activity Endo S2 and microbial transglutaminase preparations as a modular platform for dual-payload ADC construction. It reports at least 95% HPLC-SEC purity for both enzyme preparations, low host-cell-related impurities, undetected bioburden and low endotoxin specifications

Endo S2 and antibody Fc glycan remodeling

Endo S2 is an endoglycosidase capable of modifying the conserved N-linked glycans located at asparagine 297, commonly written as N297, in the Fc region of IgG antibodies.

Reference : ResearchGate

During glycan remodeling, Endo S2 can remove the heterogeneous Fc glycan while leaving a defined residual glycan structure attached to the antibody. A functionalized glycan or glycan oxazoline can then be transferred to this position.

This process creates a controlled conjugation handle at the antibody glycosylation site.

The Endo S2 approach is particularly attractive because it can:

- Target the conserved Fc glycosylation region.

- Replace heterogeneous native glycans with defined structures.

- Introduce azide, alkyne or other bioorthogonal functional groups.

- Support subsequent click-chemistry reactions.

Produce glycosite-specific antibody conjugates.

Studies have demonstrated that Endo S2 can accept chemically modified glycan oxazolines, including substrates carrying functional groups or pre-attached drug-related structures.

Microbial transglutaminase, commonly abbreviated as mTGase or mTG, catalyzes acyl-transfer reactions between suitable glutamine residues and primary amines.

In antibody engineering, mTGase can modify accessible glutamine sites or engineered glutamine-containing tags. One particularly important Fc-region site is glutamine 295, or Q295.

Depending on the antibody format and glycosylation state, mTGase can attach an amine-containing linker to Q295, creating a stable isopeptide bond. This linker can carry a payload directly or contain a functional handle for subsequent payload attachment.

The principal advantages of mTGase-mediated antibody conjugation include :

- Defined conjugation sites

- Stable covalent bond formation

- Reduced ADC heterogeneity

- Compatibility with different linker structures

- Potential control over the resulting DAR

- Applicability to multiple antibody formats

mTGase-mediated protocols have been used to produce homogeneous ADCs and have demonstrated broad compatibility with different antibodies and drug-linker structures.

Orthogonal conjugation means that two reactions address different molecular sites without substantially interfering with one another.

In the Endo S2-mTGase platform:

Although Q295 and N297 are located close to each other in the Fc region, recent research demonstrated that both positions can be modified sequentially.

In the reported system, mTGase showed strong activity toward an Endo S2-deglycosylated antibody retaining a fucosylated GlcNAc structure at N297. The study also found that Endo S2-mediated remodeling at N297 tolerated prior modification of Q295. These results support the use of the two enzymes as an orthogonal platform for introducing two distinct payloads into the Fc domain.

Step 1: Antibody preparation

The monoclonal antibody should first be characterized for:

- Identity and molecular mass

- Aggregation level

- Fc glycan composition

- Purity and integrity

- Antigen-binding activity

- Concentration and buffer compatibility

Buffer components containing reactive primary amines may interfere with transglutaminase-mediated reactions and should be evaluated before conjugation.

Step 2: Fc glycan trimming with Endo S2

Endo S2 hydrolyzes the native Fc N-glycan at N297.

The resulting antibody contains a more uniform residual glycan structure and can be purified or directly processed, depending on the validated workflow.

Important reaction variables include:

- Enzyme-to-antibody ratio

- Reaction temperature

- Buffer composition and pH

- Incubation time

- Initial glycan profile

- Antibody concentration

Step 3: mTGase-mediated linker installation

Microbial transglutaminase attaches an amine-functionalized linker to the accessible Q295 position.

The linker may contain:

- The first payload

- An azide group

- An alkyne group

- Tetrazine or trans-cyclooctene functionality

- Another bioorthogonal reactive handle

Reaction completion should be confirmed by intact-mass analysis, peptide mapping or another site-specific analytical method.

Step 4: Endo S2-mediated glycan remodeling

A functionalized glycan oxazoline is transferred to the residual glycan at N297.

The introduced glycan can contain a second orthogonal handle that is chemically distinguishable from the functionality installed through mTGase.

Step 5: Attachment of the two payloads

Each payload is attached through a selective reaction corresponding to its functional handle.

For example:

1. Azide-DBCO strain-promoted cycloaddition
2. Tetrazine-trans-cyclooctene ligation
3. Thiol-maleimide coupling
4. Enzyme-compatible amide-bond formation

The two chemistries must be mutually compatible to prevent cross-reaction, payload scrambling or incomplete conjugation.

Step 6: Purification and characterization

The final dual-payload ADC is purified to remove:

- Residual enzymes

- Free payloads

- Unreacted linkers

- Glycan donors

- Reaction by-products

- Aggregated antibody species

Size-exclusion chromatography, ion-exchange chromatography or hydrophobic-interaction chromatography may be used depending on the physicochemical characteristics of the ADC.

A dual-payload ADC requires more extensive characterization than a conventional single-payload conjugate.

Identity and payload distribution

Intact or reduced LC–MS can confirm the expected molecular-mass increase and determine whether both payloads are present.

Peptide mapping should be used to confirm modification at the intended Q295 and N297-associated sites.

Drug-to-antibody ratio

For dual-payload ADCs, one total DAR value may be insufficient. The analysis should ideally report:

1. DAR of payload A
2. DAR of payload B
3. Combined DAR
4. Distribution of partially conjugated species
5. Percentage of unconjugated antibody

A construct described as DAR 4 may represent several different molecular populations unless the contribution of each payload is reported separately.

Aggregation

HPLC-SEC is commonly used to measure monomer, high-molecular-weight species and fragmentation.

Payload hydrophobicity can increase aggregation risk, particularly when two distinct payload systems are installed on the same antibody.

Charge heterogeneity

Ion-exchange chromatography or capillary isoelectric focusing can identify charge changes caused by glycan remodeling, linker installation or payload conjugation.

Glycan characterization

Hydrophilic-interaction chromatography, mass spectrometry or glycopeptide mapping can confirm:

1. Removal of the original Fc glycans
2. Presence of the residual glycan acceptor
3. Transfer of the functionalized glycan
4. Glycan occupancy at N297
5. Absence of unintended glycoforms

Biological activity

Characterization should include:

1. Antigen-binding affinity
2. Target-dependent internalization
3. Cytotoxicity of each single-payload control
4. Cytotoxicity of the dual-payload ADC
5. Bystander-killing activity
6. Activity in resistant cell models
7. Serum or plasma stability
8. Payload-release kinetics

Two independently addressable Fc sites :

N297 glycan remodeling and Q295 transglutamination provide two chemically distinguishable conjugation positions.

Improved molecular homogeneity :

Defined attachment sites can reduce the number of molecular variants compared with stochastic lysine or cysteine conjugation.

Controlled payload placement :

The platform allows payload A and payload B to be assigned to specific Fc sites rather than distributed randomly across the antibody.

Flexible payload combinations :

Different cytotoxic drugs, imaging agents, immune stimulants or targeted degraders may potentially be combined, provided their linkers and reaction conditions are compatible.

Potentially scalable enzymatic processing :

Enzymatic reactions generally operate under aqueous and relatively mild conditions. High-purity, high-activity enzymes may also reduce enzyme loading and simplify downstream clearance, although actual scalability must be confirmed for each process.

Preservation of the antigen-binding region :

Both described modification sites are situated in the Fc region, away from the antigen-binding variable domains. Nevertheless, binding activity should always be verified after conjugation.

Selecting a rational payload pair

Two payloads should not be combined solely because they can be chemically attached. The pair should have a defensible biological rationale.

Relevant considerations include:

1. Different mechanisms of action
2. Activity against distinct resistance pathways
3. Complementary membrane permeability
4. Compatible intracellular release
5. Non-overlapping toxicity profiles
6. Similar effective concentration ranges

A strongly imbalanced combination may behave functionally as a single-payload ADC.

Controlling payload ratio

The ratio of payload A to payload B can influence efficacy, pharmacokinetics, aggregation and toxicity.

Each conjugation step therefore requires independent control of reaction conversion and site occupancy.

Managing hydrophobicity

Installing two hydrophobic payload systems may reduce solubility and increase aggregation or nonspecific clearance.

Potential mitigation approaches include hydrophilic spacers, optimized PEG length, polar linker components and lower total DAR.

Preserving enzyme activity

The order of the Endo S2 and mTGase reactions can influence conversion. Buffer composition, residual glycans, prior Fc modification and steric effects may change enzyme accessibility.

The optimal reaction sequence must therefore be established experimentally rather than assumed to be interchangeable.

Demonstrating enzyme clearance

For manufacturing-oriented workflows, downstream processing must demonstrate adequate removal of both enzymes and other process-related impurities.

Residual-enzyme assays may need to be developed specifically for Endo S2 and mTGase.

Regulatory and CMC complexity

A dual-payload ADC introduces more critical quality attributes than a conventional ADC. Each payload, linker, attachment site and partially conjugated species may affect the final safety and efficacy profile.

Claims of GMP suitability should consequently be supported by manufacturing documentation, traceability, analytical validation and lot-specific certificates rather than enzyme purity alone.

What is a dual-payload ADC?

=> A dual-payload ADC is an antibody conjugated with two different therapeutic payloads. The payloads are selected to provide complementary mechanisms of action or activity against heterogeneous and resistant tumor-cell populations.

What does Endo S2 do in ADC manufacturing?

=> Endo S2 remodels the conserved Fc glycan at N297. It can remove heterogeneous native glycans and facilitate installation of a defined, functionalized glycan carrying a conjugation handle.

What does microbial transglutaminase do?

=> Microbial transglutaminase catalyzes covalent attachment of an amine-containing linker to a suitable glutamine residue. In suitable IgG substrates, the Fc Q295 residue can be used for site-specific conjugation.

Can Endo S2 and mTGase be used on the same antibody?

=> Yes. Research published in 2026 demonstrated orthogonal modification of the Q295 and N297 regions using mTGase and Endo S2, respectively, enabling installation of two different ligands or payloads.

Are dual-payload ADCs more effective than single-payload ADCs?

=> They can be more effective in selected biological models, particularly where antigen heterogeneity or resistance to one payload limits treatment. However, efficacy depends on the target, linker chemistry, payload combination, DAR and tumor biology. Preclinical superiority does not guarantee clinical superiority.

Does site-specific conjugation guarantee a homogeneous ADC?

=> It can substantially improve homogeneity, but the final preparation may still contain incomplete conjugates, aggregates, glycan variants, linker-related impurities and payload hydrolysis products. Comprehensive analytical characterization remains necessary.

What enzyme quality attributes are important?

=> Important attributes include enzyme identity, specific activity, purity, host-cell protein, host-cell DNA, endotoxin, bioburden, formulation stability and batch consistency. For manufacturing applications, residual-enzyme clearance and supply-chain traceability are also important.

The combination of Endo S2-mediated Fc glycan remodeling and microbial transglutaminase-mediated Q295 conjugation provides a scientifically compelling route for constructing site-specific dual-payload antibody–drug conjugates.

The platform offers two independently addressable sites within the Fc region, supporting controlled payload placement and improved molecular definition. This may help researchers design ADCs capable of targeting heterogeneous tumor populations and reducing dependence on a single cytotoxic mechanism.

Nevertheless, successful development requires more than high conjugation conversion. Payload compatibility, individual and combined DAR, aggregation, linker stability, enzyme clearance, biological activity and process reproducibility must all be carefully optimized.

As dual-payload ADC development progresses, orthogonal enzymatic conjugation is positioned to become an important enabling technology for producing structurally defined next-generation antibody therapeutics.

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2. Yamazaki CM, et al. Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance. Nature Communications. 2021.
3. Dickgiesser S, et al. Site-Specific Antibody-Drug Conjugation Using Microbial Transglutaminase. 2019.
4. Dickgiesser S, et al. Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminase. 2020.
5. Tang F, et al. One-pot N-glycosylation remodeling of IgG with non-natural sialylglycopeptides enables glycosite-specific and dual-payload antibody-drug conjugates. 2016.