01 — Mechanism of Action
Overview
An ADC is a guided missile: a monoclonal antibody (mAb) delivers a potent cytotoxic payload selectively to antigen-expressing tumor cells. The concept marries the specificity of immunotherapy with the killing power of chemotherapy, ideally widening the therapeutic window beyond what either modality achieves alone.
ADC Architecture
[Antibody] — [Linker] — [Payload]
| Component | Role | Critical Properties |
|---|---|---|
| Antibody (mAb) | Targeting vehicle; mediates cell binding, internalization, and half-life | High affinity for tumor antigen; minimal shedding; FcRn-mediated recycling |
| Linker | Chemical tether; controls payload release site (extracellular vs. intracellular) | Plasma stability; selective cleavage in tumor cell |
| Payload (warhead) | Cytotoxic agent | Sub-nanomolar IC₅₀ (100–1000× more potent than standard chemo); membrane permeability (governs bystander effect) |
Step-by-Step Cellular MOA
Step 1 — Target Antigen Binding
The antibody binds to a tumor-associated antigen (TAA) on the cell surface via its Fab region. Ideal TAA characteristics:
- High and homogeneous expression on tumor cells
- Low or absent expression on normal tissues
- Capacity for internalization upon antibody binding
- Minimal shedding of the ectodomain into circulation (shed antigen acts as a decoy, consuming ADC before it reaches the cell)
Examples: HER2 (ErbB2), CD30, CD33, Nectin-4, TROP-2, FRα, BCMA
Step 2 — Receptor-Mediated Internalization
After antigen binding, the ADC–antigen complex is internalized via receptor-mediated endocytosis (clathrin-coated pits or macropinocytosis). The rate of internalization varies by target and is a key determinant of intracellular payload delivery efficiency.
- Rapid internalization: CD33 (mins), CD30
- Slower internalization: HER2 (recycled via FcRn-like mechanisms in some models)
- Some ADCs trigger accelerated internalization upon binding (e.g., biparatopic HER2 ADCs)
Step 3 — Endosomal Trafficking
Internalized vesicles (early endosomes) mature into late endosomes. The luminal pH drops progressively:
- Early endosome: pH ~6.0–6.5
- Late endosome: pH ~5.5–6.0
- Lysosome: pH ~4.5–5.0
This acidification is exploited by acid-labile linkers (e.g., hydrazone) which cleave at low pH.
Step 4 — Lysosomal Processing
In the lysosome:
- Protease-cleavable linkers (e.g., Val-Cit dipeptide) are cleaved by cathepsin B and other cysteine proteases.
- Disulfide linkers are reduced by the high intracellular glutathione (GSH ~1–10 mM vs. ~2–20 µM extracellular).
- Non-cleavable linkers (thioether) resist enzymatic cleavage. The antibody itself is fully degraded by lysosomal proteases, releasing an amino acid–linker–payload catabolite (e.g., lysine-SMCC-DM1 from T-DM1/Kadcyla).
Step 5 — Payload Release and Intracellular Activity
Free payload (or catabolite) is released into the cytoplasm where it exerts its cytotoxic mechanism:
| Payload Class | Intracellular Target | ADC Example |
|---|---|---|
| Auristatins (MMAE, MMAF) | β-tubulin (microtubule depolymerization) | Adcetris, Padcev |
| Maytansinoids (DM1, DM4) | β-tubulin (microtubule depolymerization) | Kadcyla, Elahere |
| Calicheamicin | DNA (double-strand breaks, minor groove binding) | Mylotarg, Besylomab |
| PBD dimers (SG3199, tesirine) | DNA (interstrand crosslinks, minor groove) | Zynlonta |
| DXd (exatecan derivative) | Topoisomerase I (DNA replication block) | Enhertu |
| SN-38 | Topoisomerase I | Trodelvy |
Step 6 — Bystander Effect
If the released payload is membrane-permeable, it can diffuse out of the killed cell and enter neighboring cells — including antigen-negative cells. This “bystander effect” is therapeutically relevant for tumors with heterogeneous antigen expression.
| Payload | Bystander Effect | Membrane Permeability |
|---|---|---|
| MMAE (free, uncharged) | Strong | High |
| DXd | Moderate-Strong | Moderate |
| SN-38 | Moderate | Moderate |
| MMAF | Weak | Low (charged carboxyl group) |
| Lys-SMCC-DM1 (catabolite) | Minimal | Low (charged, polar) |
| Calicheamicin | Minimal | Low |
Clinical implication: Strong bystander effect is desirable for solid tumors (heterogeneous antigen expression) but may drive off-target toxicity in normal tissue adjacent to tumor.
Step 7 — Cell Death
Tumor cell death occurs via apoptosis (caspase activation), mitotic arrest (tubulin inhibitors), or DNA damage response (DSB-inducing payloads). Some payloads also trigger immunogenic cell death (ICD), potentially activating anti-tumor immunity.
Resistance Mechanisms
| Mechanism | Description | Affected ADCs |
|---|---|---|
| Antigen downregulation | Tumor cells reduce target expression under selection pressure | All |
| Reduced internalization | Loss of internalization-competent receptor isoforms | All |
| Lysosomal dysfunction | Upregulation of lysosomal drug efflux pumps (MDR1/P-gp) | Auristatin-based |
| Drug efflux (MDR1/ABCB1) | Efflux of membrane-permeable payloads | MMAE, DXd |
| Anti-apoptotic gene upregulation | BCL-2/BCL-xL overexpression | Tubulin inhibitors |
| Antigen shedding | High circulating antigen sequesters ADC | CD33, HER2 |
| Tumor heterogeneity | Antigen-negative subclones repopulate | MMAF-based (no bystander) |
Key Papers
- Chari et al. (2014) Acc Chem Res — foundational ADC mechanism review
- Ogitani et al. (2016) Clin Cancer Res — T-DXd mechanism and bystander effect characterization
- Ritchie et al. (2013) mAbs — auristatin-based ADC intracellular trafficking