What Are Antibody-Drug Conjugates
Antibody drug conjugates (ADCs) are a class of cancer therapeutics that incorporate the precise targeting ability of monoclonal antibodies (mAbs) with the potent cytotoxicity of chemotherapeutic payloads. The binding of an ADC to its target results in specific targeting of the highly toxic drug payload to the cancer cells, while minimizing off-target toxicities. Since the first FDA approval of gemtuzumab ozogamicin in 2000, ADCs have gained tremendous momentum. To date, 14 ADCs have been approved globally as of December 2021 and more than 100 ADC candidates are in clinical trials. These therapies are mostly used in hematologic malignancies and solid tumors and the list is expanding as more targets and conjugation chemistry continue to be developed.
Figure 1. A brief history of ADC drug development[1].
What Are the Essential Components of an Antibody-Drug Conjugate?
ADC is composed of antibody, cytotoxic payload and chemical linker. An ideal ADC is considered to be stable in the blood circulation, accumulate at the therapeutic site with high specificity and release cytotoxic payload around the target (e.g. cancer cells).
Figure 2. The structure of an ADC.
How Is the Target Antigen Selected for ADC Design?
The appropriate selection of the target antigen for an ADC is critical for both efficacy and safety. The ideal target is a transmembrane protein that is overexpressed on tumor cells and is minimally expressed in normal tissues. It should allow for efficient internalization of the ADC-antigen complex and not be secreted. Clinically validated targets include HER2, TROP2, CD19, CD22, and CD33. More recent investigations have suggested that ADCs could be targeted against stromal and vascular elements of the tumor microenvironment which are more genetically stable and broadly expressed across tumor types.
Figure 3. Target antigens for ADCs in solid tumors[2].
How Do Antibodies Contribute to ADC Selectivity and Function?
The monoclonal antibody component of an ADC acts as a delivery vehicle, which is directed against the target antigen. The predominant isotype used is IgG1 due to its long half-life, strong Fc-mediated immune effector functions (ADCC, ADCP, CDC), and stable structure. Affinity should be optimized, as higher affinity enhances internalization but limits deep penetration due to the "binding site barrier". Full-length IgGs (~150 kDa) have longer half-lives but limited tissue penetration in solid tumors. To improve this, smaller engineered fragments are being explored; however, these tend to have reduced half-life and may need additional molecular engineering to improve systemic stability.
What Is the Role of Linkers in ADC Stability and Drug Release?
The linker is the part of the ADC that connects the antibody and the cytotoxic payload. The choice of linker dictates the stability of the ADC in circulation and the drug release site. Linkers can be cleavable or non-cleavable:
- Cleavable linkers allow drug release in tumor specific conditions (e.g. pH, enzyme, glutathione). Acid-labile hydrazones, disulfides and protease cleavable peptide (e.g. valine-citrulline) linkages are typical.
- Non-cleavable linkers (e.g. thioether or maleimidocaproyl, such as in ado-trastuzumab emtansine) are only released when the antibody is degraded in the lysosome.
Each type has trade-offs between systemic stability and efficient intracellular release.
Alfa Chemistry offers a broad portfolio to release the payload under specific intracellular conditions, ensuring an optimal therapeutic window and minimizing off-target effects.
Alfa Chemistry also offers innovative payload-linker architectures which covalently combine the cytotoxic payload and the linker into one compound ready for conjugation. This approach reduces the ADC development time and increases the uniformity and stability of the conjugate.
Which Cytotoxic Payloads Are Used in ADCs and Why?
Due to low tumor accumulation (~2%), ADCs utilize highly potent cytotoxins. Here are some examples of small-molecule payloads used in ADC drugs:
Table 1 The representative small-molecule payloads used in ADC drugs[1]
| Categories | Names | Mechanisms | Potency (IC50 or EC50) |
| Tubulin inhibitors | Auristatins | Promote tubulin polymerization and target at the β-subunits of tubulin dimer to perturb microtubule growth | 0.05–0.1 nM |
| Maytansinoids | Block the polymerization of tubulin dimer and inhibit the formation of mature microtubules | 0.05–0.1 nM | |
| Tubulysins | Inhibit tubulin polymerization | 0.1–1 nM | |
| DNA damaging agents | Calicheamicins | DNA double strand break: bind with DNA in the minor groove and cause strand scission | 0.1–1 nM |
| Duocarmycins | DNA alkylation: bind to the minor groove of DNA and alkylate the nucleobase adenine at the N3 position | 1–10 pM | |
| Exatecans | Topoisomerase I inhibitor: bind to the topoisomerase I and DNA complex and prevent DNA re-ligation and therefore causes DNA damage which results in apoptosis | 1–10 nM | |
| Pyrrolobenzodiazepines | Crosslinking of DNA: produce DNA interstrand cross-links with high efficiency in both naked DNA and in cells. | 0.1–1 pM | |
| Immunomodulators | TLR agonists | Potent stimulation of innate and adaptive immunity as well as their effects on the tumor microenvironment | ~1 μM |
| STING agonists | Promote activation of type I interferons and other inflammatory cytokines | ~100 nM |
Notably, immune-stimulating antibody conjugates (ISACs) are emerging, incorporating toll-like receptor (TLR) or STING agonists to activate innate immunity and reshape the tumor microenvironment. Several are in clinical trials, offering a promising strategy for enhancing anti-tumor efficacy.
Alfa Chemistry provides a wide range of payloads including microtubule protein inhibitors, DNA damaging agents and other novel cytotoxic agents for ADC development and preclinical research.
How Are Antibody-Drug Conjugates Synthesized and Conjugated?
What Conjugation Methods Are Used to Assemble ADCs?
Conjugation determines the drug-to-antibody ratio (DAR), a critical quality attribute. Early ADCs employed random conjugation through:
- Lysine residues: Amide bonds formed with activated esters; leads to heterogeneous DAR (0–8).
- Cysteine residues: Reduction of interchain disulfides exposes reactive thiols and produces more consistent DAR (2, 4, 6).
While widely used, random conjugation introduces variability in stability, potency, and manufacturability. Modern strategies favor site-specific conjugation to enhance product consistency and pharmacological behavior.
Figure 4. Conjugation strategies for ADC development, which can be prepared by traditional random conjugation methods (on exposed lysine residues or reduced interchain cysteine residues) or by site-specific conjugation methods such as sugar conjugation[3].
How Is Site-Specific Conjugation Achieved?
Advanced site-specific conjugation approaches include:
- Engineered cysteines: Introduce unique thiols at predetermined sites.
- Disulfide rebridging: Use bifunctional reagents to maintain antibody structure while enabling payload attachment.
- Non-natural amino acids: Incorporate reactive groups (e.g., azides, alkynes) for orthogonal chemistry; enable click reactions.
- Enzymatic conjugation: Employ transglutaminase or formylglycine-generating enzymes to target engineered recognition motifs.
- Glycan remodeling: Modify Fc N-linked glycans at Asn297 to attach payloads without disrupting antigen binding.
- pClick chemistry: Utilize proximity-activated crosslinkers to conjugate azido-functionalized peptides to lysine residues with no need for antibody engineering.
How Do Antibody-Drug Conjugates Exert Cytotoxic Effects in Cancer Cells?
Upon systemic administration, ADCs bind to their cognate antigen on tumor cells, followed by internalization and trafficking through endosomes and lysosomes. Inside the acidic or enzyme-rich lysosomal environment, the linker is cleaved (if cleavable) or the antibody degraded (if non-cleavable), releasing the active drug. The cytotoxic payload induces cell death via apoptosis, mitotic arrest, or DNA damage.
Figure 5. Mechanism of action of ADC: After ADC binds to the target antigen, the ADC-target complex is internalized (endocytosis) and subsequently degraded by lysosomes[4].
ADCs can also exhibit bystander killing, where the released drug diffuses to adjacent antigen-negative tumor cells. This is especially useful in heterogeneous tumors. Additionally, ADCs leverage the antibody's Fc region to induce:
- ADCC: Engagement of NK cells via FcγRIII.
- ADCP: Macrophage-mediated phagocytosis.
- CDC: Activation of the complement cascade.
Some ADCs, such as T-DM1, inhibit receptor signaling pathways directly—e.g., blocking HER2 dimerization and downstream PI3K/MAPK signaling, leading to apoptosis.
FAQs About Antibody-Drug Conjugates
1. What is the average drug-to-antibody ratio (DAR) considered optimal?
Most approved ADCs have DARs between 2 and 4 to balance potency, stability, and pharmacokinetics.
2. Are all ADC payloads membrane-permeable?
No. MMAE is membrane-permeable and can produce bystander effects, while MMAF is non-permeable and acts only intracellularly.
3. What makes ISACs different from traditional ADCs?
ISACs use immunomodulators as payloads to stimulate innate or adaptive immunity in addition to direct cytotoxicity.
4. Do ISACs fall under the same regulatory framework as ADCs?
ISACs are a subclass of ADCs, but their immune-modulatory effects may require additional safety assessments during development.
5. Can ADCs be used for non-oncologic indications?
While still under investigation, ADCs hold potential for targeting pathogenic cells in autoimmune diseases or infections.
6. How does glycan remodeling improve ADC development?
It allows conjugation away from the antigen-binding site, preserving antibody function and reducing aggregation.
7. What are the main regulatory challenges in ADC development?
Controlling DAR homogeneity, linker stability, and systemic toxicity are primary concerns in regulatory approval.
8. How is ADC immunogenicity managed?
By using fully humanized antibodies and minimizing non-natural residues, immunogenicity risks are significantly reduced.
9. How stable are ADCs in plasma?
Linker chemistry and conjugation strategy influence plasma stability. Non-cleavable linkers generally show higher in vivo stability.
References
- Li M., et al. Antibody-Drug Conjugate Overview: a State-of-the-art Manufacturing Process and Control Strategy. Pharmaceutical Research. 2024, 41(3), 1-22.
- Diamantis N., et al. Antibody-drug conjugates--an emerging class of cancer treatment. Br J Cancer. 2016, 114(4), 362-367.
- Metrangolo V., et al. Antibody-Drug Conjugates: The Dynamic Evolution from Conventional to Next-Generation Constructs. Pharmaceutical Research. 2024, 16(2), 447.
- Byun J. H., et al. RECEPTOR-MEDIATED ENDOCYTOSIS MODELING OF ANTIBODY-DRUG CONJUGATES TO THE RELEASED PAYLOAD WITHIN THE INTRACELLULAR SPACE CONSIDERING TARGET ANTIGEN EXPRESSION LEVELS. Journal of Applied Analysis & Computation. 2020, 10(5), 1848-1868.