Background

Antibody-drug conjugates (ADCs) represent one of the fastest-growing classes of oncology therapeutics, with 14 approved drugs and a market projected to exceed $30 billion by 2030. However, traditional chemical conjugation methods produce heterogeneous mixtures with variable drug-to-antibody ratios (DAR), compromising efficacy and safety. Enzymatic precision — leveraging glycan remodeling enzymes, sortases, and transglutaminases — enables site-specific ADC conjugation with defined DAR and improved therapeutic index. Hzymes provides GMP-grade EndoS2, EndoS2-pro, and mTGase for ADC bioconjugation, supporting your development from research through commercial manufacturing.

Marketed ADC Drugs

As of the first half of 2025, 19 ADC drugs have been approved globally, including 3 independently developed by domestic enterprises. At the 2025 World Conference on Lung Cancer (WCLC), Baili Tianheng’s EGFR/HER3 bispecific ADC study showed a first-line combination with osimertinib achieving an objective response rate (ORR) of 100%, while monotherapy in later lines of treatment demonstrated a median progression-free survival (mPFS) exceeding 12 months. Henlius’ PD-L1 IO+ADC in non-squamous NSCLC patients in third-line or later treatment achieved an ORR of 46.2% and a disease control rate (DCR) as high as 96.2%.

Currently, about 230 ADC drugs are in clinical development worldwide, spanning Phase I to Phase III, with targets primarily focused on HER2, TROP2, EGFR, Nectin-4, and Claudin18.2. At AACR and WCLC 2025, new ADC targets also emerged, including B7H3, DLL3, CDH17, MLSN, PSMA, and CEACAM5.

ADC Structure and Function

An antibody-drug conjugate (ADC) links a monoclonal antibody to a small-molecule cytotoxin, forming a therapeutic composed of three main parts: monoclonal antibody (mAb), linker, and payload. Mechanistically, after administration, the ADC specifically recognizes tumor-associated antigens via its antibody component, undergoes endocytosis into tumor cells, and is degraded in lysosomes to release potent cytotoxic payloads. This process kills tumor cells and can trigger a “bystander effect.”

Characteristics of ADC Components

Monoclonal Antibody (mAb): Effective antigen binding

Target antigens are highly expressed and specific on tumor cells while being low or absent in normal tissues. This ensures high specificity binding and reduces “off-target” effects.

Linker: Stability and efficient cleavage

Maintain stability in blood circulation to avoid off-target toxicity. Upon delivery into target cells, it enables efficient cleavage and rapid payload release.

Payload: High potency and efficacy

Common types include microtubule inhibitors (maytansinoids such as DM1, DM4, MMAE, MMAF), DNA topoisomerase I inhibitors (e.g., camptothecin derivatives SN-38, Dxd), immune modulators (TLR agonists), apoptosis inducers (Bcl-xl inhibitors), RNA polymerase inhibitors, transcription inhibitors, and protease inhibitors. Since only about 2% of intravenously administered ADCs reach tumor sites, highly potent payloads (IC50 in the nM to pM range) are essential.

Conjugation: Uniform Drug-to-Antibody Ratio (DAR)

The conjugation site and method affect ADC stability, efficacy, and pharmacokinetics. Achieving DAR uniformity enhances drug stability and reduces toxicity.

Conjugation Methods

Chemical Conjugation vs. Enzymatic Conjugation

With the advancement of ADC technologies, site-specific conjugation has become the preferred strategy for ADC development. Among disclosed ADCs, site-specific conjugation accounts for 445 (77%), whereas non-specific conjugation accounts for only 131 (23%). Since the first site-specific ADC entered clinical trials in 2010, its proportion in clinical development has steadily increased, reaching 28 molecules by 2024.

Chemical Conjugation

Chemical conjugation is based on the non-selective modification of amino acid residues naturally present in antibodies, such as lysine and cysteine conjugation.

Enzymatic Site-Specific Conjugation

No.1 Glycan Conjugation (Endo S) – Two Enzymes, Three Steps

Endo S, an endoglycosidase, specifically hydrolyzes the glycosidic bond between the core chitobiose (GlcNAc-GlcNAc) of glycans, exposing a terminal GlcNAc residue. A glycosyltransferase (GalT/Y289L) is then used to attach an azide-containing substrate (UDP-GalNAz) to the GlcNAc. Finally, click chemistry (SPAAC or CuAAC) links the payload bearing the complementary group (e.g., DBCO or alkyne).

Hzymes Endo S (Cat#HBP000126): Sequence derived from Streptococcus pyogenes, recombinantly expressed in E. coli. It is of high purity, high activity, thermostable, free of exoglycosidase contamination, and lacks proteolytic activity.

No.2 Glycan Conjugation (Endo S2) – One Enzyme, Two Steps

Endo S2 hydrolyzes the β-1,4 glycosidic bond within the chitobiose core of N-glycans on the Fc region of IgG, leaving a core-fucosylated GlcNAc at N297. Endo S2 also possesses transglycosylation activity, enabling simultaneous transfer of an azide-containing substrate (LacNAc) onto GlcNAc, thus achieving glycan conjugation in a single step.

Hzymes Endo S2 Pro (Cat#HBP000133): Developed using the Hevo AI+ platform for conserved site analysis, de novo design tools, and auxiliary modeling to address higher-order effects. Structural analysis identified key residues, improving internal hydrophobicity and thermal stability.

Recombinantly expressed in E. coli and validated with trastuzumab, mass spectrometry confirmed Endo S2 Pro thoroughly cleaves antibody glycans and achieves complete LacNAc conjugation.

No.3 Transglutaminase Conjugation (mTGase) – One Enzyme, One Step

Microbial transglutaminase (mTGase) catalyzes the formation of peptide bonds between the γ-carboxamide group of glutamine (Gln) residues and primary amines. It enables covalent conjugation of linker-payload molecules to specific glutamine residues (e.g., Q295) of antibodies, producing DAR values of 2 or 4.

Hzymes mTGase (Cat#HBP000132): Sequence derived from Streptoverticillium mobaraense, recombinantly expressed in E. coli. Catalyzes acyl transfer of γ-carboxamide groups from glutamine residues to primary amines. Unlike native microbial TGase, recombinant mTGase does not require Ca²⁺ for catalytic activity. It specifically recognizes the LLQG glutamine motif.

No.4 Sortase A Conjugation – One Enzyme, One Step

Sortase A recognizes the specific pentapeptide sequence LPXTG (where X is any amino acid), cleaves the peptide bond between threonine (T) and glycine (G), and forms an acyl-enzyme intermediate. This intermediate is then attacked by an oligo-glycine nucleophile, forming a new peptide bond (LPXT-Gly(n)-Payload).

Hzymes Sortase A (Cat#HBP000122): Derived from Staphylococcus aureus, recombinantly expressed in E. coli. Specifically recognizes C-terminal conserved motifs LPETG and N-terminal GGG-XXX.

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