Traditional ADCs: The Need for Precision
Over the past decade, all FDA-approved antibody-drug conjugates (ADCs) have been heterogeneous mixtures composed of monoclonal antibodies (mAbs) conjugated with varying numbers of cytotoxic drugs at multiple, random sites. However, the biopharmaceutical industry has been actively developing innovative site-specific conjugation strategies aimed at controlling both the position and number of payloads attached, while preserving structural integrity and improving product homogeneity.
Classification of Conjugation Strategies
Currently, ADC conjugation technologies can be broadly categorized into two types:
Non-Specific (Conventional) Conjugation: These techniques utilize naturally reactive amino acid residues in antibodies, such as lysine side-chain amines or thiol groups derived from reduced interchain disulfide bonds.
Site-Specific Conjugation: These advanced approaches involve engineering specific reactive groups into defined antibody locations using genetic modification, chemical modification, or enzymatic processing. Prominent examples include engineered cysteine conjugation (THIOMAB), incorporation of non-natural amino acids, enzyme-mediated coupling, and glycan-directed conjugation.
| Strategy | Non-Site-Specific Conjugation | Site-Specific Conjugation | |||||
| Method | Lysine residue | Semi-reductive alkylation of lysine | Modified active semi-reductive alkylation | Disulfide rebridging | Unnatural amino acid | Enzyme-assisted conjugation | Cysteine residue mutation |
| Advantages | Fast and convenient | Relatively high uniformity, suitable for acidic-sensitive drugs | High uniformity, tunable DAR and hydrophobicity | High uniformity, minimal impact on antibody structure | High uniformity, tunable DAR, stable linkage | High uniformity, high DAR efficiency, controllable drug loading | High uniformity, stable disulfide sequence |
| Disadvantages | Heterogeneity low DAR (0–8) Poor stability Narrow therapeutic window |
Unstable conjugation structure Over-reduction reduces effective payload |
Requires protein engineering DAR 2 limitation |
Limited bridge compatibility DAR 4 limitation |
Requires protein engineering Unnatural amino acids may affect expression and folding Immunogenicity risk |
Requires protein engineering External sequence or tag may cause immunogenicity |
Stability affected by redox conditions in tumor microenvironment |
First-Generation ADC Technology: Random Conjugation
Traditional ADCs typically involve non-specific conjugation to lysine residues or reduced interchain cysteines. A single IgG molecule contains 80-90 lysines, with conjugation potentially occurring on nearly 40 different residues. Similarly, disulfide bond reduction generates multiple reactive thiols, often compromising antibody integrity. As a result, early ADCs were highly heterogeneous, exhibited poor stability, and suffered from narrow therapeutic windows and unpredictable pharmacokinetics.
Second-Generation ADC Technology: Site-Specific Conjugation
To overcome the limitations of first-generation ADCs, newer strategies involve introducing bio-orthogonal functional groups at precise locations within the antibody. Although effective, such modifications may sometimes alter antibody folding, stability, or biological function.
1. Engineered Cysteine Conjugation (THIOMAB Technology)
Genentech pioneered the THIOMAB approach, which introduces engineered cysteine residues into specific antibody sites via genetic engineering. Under reducing conditions, these cysteines and native disulfides are reduced, then reoxidized using CuSO4 to reform disulfide bonds, allowing site-specific drug conjugation.
Key benefits of THIOMAB ADCs include:
- High homogeneity (consistent Drug-Antibody Ratio, typically DAR 2)
- Preserved antigen-binding and antibody structure
- Enhanced in vivo antitumor efficacy and tolerability
- Reduced systemic toxicity
2. Incorporation of Non-Natural Amino Acids (nnAAs)
This method involves inserting non-natural amino acids into the antibody’s sequence, thereby creating chemically reactive handles on the antibody surface. These unique residues enable:
- Precise site control
- Consistent DAR values
- Flexibility in engineering DAR to specific values
Common nnAAs used include:
- p-acetylphenylalanine
- p-azidomethyl-L-phenylalanine
- Azidolysine
These contain reactive ketone or azide groups that facilitate efficient, uniform drug conjugation.
Drawbacks:
- Requires genetic engineering
- May reduce antibody expression yields
- Can trigger immunogenic responses due to non-native sequences
- Increased risk of aggregation from hydrophobic residues
3. Enzyme-Mediated Site-Specific Conjugation
Enzymatic conjugation uses enzymes to facilitate targeted coupling between the antibody and payload. This method offers:
- High precision
- Reduced heterogeneity
- Improved stability
However, introducing enzyme-recognized sequences may also increase immunogenic potential. Therefore, extensive immunogenicity assessment is essential during ADC development to ensure clinical safety.
Third-Generation ADC Conjugation Technology: Site-Specific Antibody Conjugation
Overcoming the Challenges of Antibody Engineering
While second-generation ADC technologies introduced site-specific conjugation, they often required extensive antibody engineering, potentially altering antibody structure or function. To overcome these limitations, researchers have shifted toward site-selective conjugation strategies that do not compromise the native integrity of antibodies. This evolution gave rise to third-generation ADC conjugation methods, which leverage unique amino acid sites or proximity-induced effects to achieve precise and efficient drug attachment.
Third-generation technologies can be broadly categorized into four main types:
- Disulfide Re-bridging
- Glycan-Based Conjugation
- Chemoselective Modification
- Proximity-Induced Conjugation
1. Disulfide Re-bridging: Controlled Payload Installation
Disulfide re-bridging involves selective reduction of the four interchain disulfide bonds in IgG1 antibodies using cross-linking reagents like TECP or DTT. Bifunctional bridging agents are then used to reconnect peptide chains while simultaneously installing payloads or introducing chemical handles.
Advantages:
- High homogeneity
- Preserves antibody structure
- Compatible with native amino acid sequences and glycosylation
Limitations:
- Risk of intrachain mis-bridging
- Typically results in a DAR (Drug-to-Antibody Ratio) of 4
Common bridging chemistries include:
- Mono-/Bis-sulfones
- 3,4-Disubstituted Maleimides
- Dibromopyridazinediones
- Divinylpyrimidines
2. Glycan-Based ADC Conjugation
IgG antibodies contain two conserved N-linked glycosylation sites at Asn297 in the CH2 domain-away from antigen-binding sites-making them ideal for conjugation without affecting binding affinity.
This method avoids genetic engineering and preserves the antibody’s natural sequence and structure.
2.1 Oxidative Glycan Modification
Selective oxidation (e.g., with periodate) of Fc glycans introduces aldehydes that can react with hydrazide-functionalized toxins to form stable hydrazone linkages. While historically used in early ADCs like gemtuzumab ozogamicin, this method risked damaging antibodies during oxidation, prompting a shift to lysine-based random conjugation.
2.2 Metabolic Glycoengineering
In this approach, culture media is supplemented with fucose analogs (e.g., 6-thioacetyl-fucose), which are metabolically incorporated into antibody glycans. These thiol groups then serve as anchors for maleimide-based payload conjugation.
2.3 Enzymatic Glycoengineering
Glycosyltransferases introduce reactive groups into antibody glycans for downstream site-specific conjugation. Multiple companies offer proprietary enzymatic glycan conjugation platforms.
3. Proximity-Induced Site-Specific Conjugation
This emerging approach exploits the spatial proximity between conjugation probes and native antibody residues (e.g., lysine, cysteine, serine) to drive selective reactivity. Without the need for genetic engineering, proximity-driven reactions allow precise conjugation at functional “hotspots” with minimal structural disruption.
Ongoing research focuses on:
Developing novel proximity-based chemistries
Identifying conjugation sites that preserve antibody functionality and pharmacokinetics
4. Other Innovative Site-Specific Conjugation Strategies
While lysine and cysteine have traditionally been used as primary conjugation targets, their inherent heterogeneity limits therapeutic performance. As a result, researchers are now exploring alternative amino acids with unique chemical reactivity, such as:
4.1 Methionine Conjugation (ReACT)
Methionine is a low-abundance amino acid (~1.8%) involved in oxidative stress defense, making it a promising target for selective modification. The ReACT method enables conjugation via methionine residues with minimal impact on protein function. However, methionine-conjugated ADCs have shown limited stability and require further optimization.
4.2 Tyrosine Conjugation
Tyrosine (3.3% natural abundance) possesses a phenol side chain that enables selective, dual-orthogonal chemical modification. Despite being partially buried in many protein structures, tyrosine can engage in hydrogen bonding and redox reactions, forming tyrosyl radicals for further modification. Common reactions include:
- Nitration
- Oxidation
- Cross-linking
- Halogenation
- AMPylation
- Glycosylation
Future Outlook: A Bright Path Ahead for ADCs
Driven by continuous advances in protein engineering, bioorthogonal chemistry, and analytical technologies, ADC conjugation strategies are becoming more sophisticated. Third-generation methods deliver superior homogeneity, stability, and targeting precision, offering significant improvements in both in vitro and in vivo efficacy.
Although many of these cutting-edge technologies remain in preclinical or early clinical stages, the promising results so far are ushering in a new generation of highly selective, safer, and more effective antibody-drug conjugates.