03 — Payload Types
Overview
ADC payloads (warheads) must be highly potent because only a fraction of the injected dose reaches the tumor cell interior. Typical payload potency requirement: IC₅₀ in the picomolar to low nanomolar range (100–1000× more potent than conventional chemotherapy). Three major classes dominate approved ADCs: microtubule inhibitors, DNA-damaging agents, and topoisomerase I inhibitors.
1. Microtubule Inhibitors
1a. Auristatins (MMAE, MMAF)
Synthetic derivatives of dolastatin 10, isolated from the sea hare Dolabella auricularia .
MMAE — Monomethyl Auristatin E
Property Value Full name Monomethyl auristatin E Molecular weight ~718 Da Mechanism Binds β-tubulin at the Vinca alkaloid binding site; prevents polymerization → mitotic arrest → apoptosis IC₅₀ (cell line) 0.1–10 nM (free drug); pM–nM when ADC-delivered Membrane permeability High (neutral at physiological pH) → strong bystander effect Solubility Moderate; hydrophobic — high DAR causes ADC aggregation and fast clearance Conjugation Via C-terminal primary amine with linker Approved ADCs Brentuximab vedotin (Adcetris), polatuzumab vedotin (Polivy), enfortumab vedotin (Padcev), tisotumab vedotin (Tivdak)
Key point : MMAE’s membrane permeability drives a potent bystander effect — critical for CD30+ lymphomas (heterogeneous antigen), Nectin-4+ urothelial cancers, and CD79b+ B-cell lymphoma.
MMAF — Monomethyl Auristatin F
Property Value Difference from MMAE C-terminal phenylalanine (carboxyl group) renders it charged at pH 7 Membrane permeability Low — charged, cannot efficiently diffuse across membranes Bystander effect Minimal Intracellular accumulation Excellent (charged catabolite trapped inside cell) Approved ADCs Belantamab mafodotin (Blenrep); glofitamab-targeting ADCs in development Clinical use BCMA-targeted myeloma (homogeneous antigen expression — bystander not required)
1b. Maytansinoids (DM1, DM4)
Natural products from Maytenus ovatus (maytansine); semi-synthetic derivatives used in ADCs.
Property DM1 DM4 Mechanism Binds β-tubulin near Vinca site; inhibits polymerization Same IC₅₀ ~0.1 nM ~0.1 nM Potency vs. vincristine ~100–1000× more potent Same Thiol group C3 free thiol → disulfide or thioether linker attachment C3 free thiol → sulfo-SPDB disulfide Linker used SMCC (non-cleavable thioether) → DM1 catabolite = Lys-SMCC-DM1 sulfo-SPDB (cleavable disulfide) → free DM4 released Approved ADCs T-DM1 (Kadcyla) — HER2+ breast cancer Mirvetuximab soravtansine (Elahere) — FRα+ ovarian Bystander effect Minimal (T-DM1: charged catabolite) Moderate (DM4 more membrane-permeable) DAR ~3.5 (T-DM1, stochastic Lys conjugation) ~3.5
T-DM1 (Kadcyla) clinical note : Non-cleavable linker means T-DM1 catabolite (Lys-SMCC-DM1) is pharmacologically active but membrane-impermeable. Very clean PK: TAb ≈ cAb at early time points; payload loss reflects DAR heterogeneity catabolism, not premature linker cleavage.
2. DNA-Damaging Agents
2a. Calicheamicin
Originally isolated from Micromonospora echinospora ssp. calichensis .
Property Value Mechanism Sequence-specific DNA minor groove binding → enediyne cycloaromatization → double-strand breaks (Bergman cyclization) Potency Extraordinarily potent: IC₅₀ ~pM; ~1000× more potent than doxorubicin Reactive species Biradical intermediate (from Bergman cyclization) causes direct DSB Membrane permeability Moderate Bystander effect Low-moderate Linker type Acid-labile hydrazone (Mylotarg/Besylomab) Approved ADCs Gemtuzumab ozogamicin (Mylotarg) — CD33+ AML; inotuzumab ozogamicin (Besylomab) — CD22+ B-ALL Toxicity concern Hepatotoxicity (veno-occlusive disease, VOD/SOS) — attributed to both premature hydrolysis and liver CD33 expression
2b. Pyrrolobenzodiazepines (PBDs)
Semi-synthetic natural product class (based on anthramycin).
Property Value Mechanism Covalent DNA minor groove binding → interstrand crosslinks → replication block → apoptosis Potency Sub-picomolar IC₅₀; among the most potent cytotoxic agents known Key feature PBD dimers (two PBD units connected by a linker) form interstrand crosslinks — more potent than monomers Cell cycle dependency Cell-cycle independent — effective against quiescent (G0) tumor cells Membrane permeability Low-moderate Approved ADC Loncastuximab tesirine (Zynlonta) — CD19+ DLBCL; uses SG3249/SG3199 PBD dimer via Val-Ala dipeptide linker Development ADCs Rovalpituzumab tesirine (DLL3+, SCLC) — discontinued due to toxicity; camidanlumab tesirine (CD25+) Toxicity Skin, pleural effusion, pericardial effusion — on-target bystander effects in normal tissues
2c. Duocarmycins (CC-1065 Analogues)
Property Value Mechanism Sequence-specific DNA alkylation in the minor groove at N3 of adenine → strand scission Potency pM IC₅₀ Prodrug activation Require intracellular prodrug activation or specific linker design Clinical status ADCs in development (e.g., SYD985/trastuzumab duocarmazine for HER2+ breast cancer — NDA submitted in EU) Bystander effect Moderate (permeable prodrug form)
3. Topoisomerase I Inhibitors
Property Value Parent drug Irinotecan (CPT-11); SN-38 is the active carboxylesterase-converted metabolite Mechanism Stabilizes Topoisomerase I – DNA cleavable complex → replication fork collision → DSBs → apoptosis Potency ~100–1000× more potent than irinotecan Membrane permeability Moderate (lactone form) Linker CL2A acid-sensitive carbonate linker (Trodelvy/sacituzumab govitecan) Approved ADC Sacituzumab govitecan (Trodelvy) — TROP-2+ TNBC, urothelial carcinoma DAR ~7.6 (high DAR; hydrophilic linker mitigates aggregation) Bystander effect Moderate-strong PK note Substantial free SN-38 released in systemic circulation due to moderate linker stability — contributes to class toxicity (diarrhea, neutropenia)
3b. DXd (Deruxtecan; Exatecan Derivative)
Property Value Parent compound Exatecan (DX-8951) — a camptothecin analogue Modification Aminomethyl substitution improves aqueous solubility vs. parent exatecan Mechanism Topoisomerase I inhibition (same mechanism as SN-38/irinotecan) Potency ~10× more potent than SN-38 in cell-free assays Membrane permeability Moderate-high (membrane-permeable at physiological pH) Linker GGFG tetrapeptide cleavable linker (cathepsin-mediated) Approved ADC Trastuzumab deruxtecan (T-DXd; Enhertu) — HER2+ BC, HER2-low BC, HER2+ NSCLC, HER2+ GC DAR ~8 (highest approved DAR; enabled by highly hydrophilic GGFG linker) Bystander effect Strong — free DXd is membrane-permeable; drives efficacy in HER2-low tumors Key toxicity Interstitial lung disease (ILD)/pneumonitis — class mechanism, requires monitoring Bioanalysis note Free DXd measurable in plasma by LC-MS/MS; T-DXd has a unique bioanalytical challenge due to high DAR and distinct catabolite profile
4. Emerging Payload Classes
4a. RNA Polymerase II Inhibitors — Alpha-Amanitin
Mechanism: Inhibits RNA Pol II → transcription arrest → apoptosis
Advantage: Active in quiescent (non-dividing) cells; MDR-independent
ADC examples: HDP-101 (BCMA; multiple myeloma) — clinical stage
Challenge: High hepatotoxicity risk; narrow therapeutic window
4b. Kinase Inhibitors
Concept: Deliver targeted inhibitors (e.g., EGFR TKI, PI3K inhibitors) via antibody to tumors expressing the kinase target
Status: Preclinical-to-early clinical; non-cleavable or acid-labile release strategies under investigation
4c. Immune Agonists (iADC)
Concept: Deliver TLR agonists or STING agonists to the tumor microenvironment via ADC
Advantage: Simultaneous tumor killing and immune activation
Status: Early clinical (e.g., BDC-1001, TLR7/8 agonist ADC targeting HER2)
Payload Property Comparison
Payload Class IC₅₀ (free) Bystander MDR sensitive Cell cycle MMAE Tubulin ~0.5 nM Strong Yes (P-gp) M phase MMAF Tubulin ~0.5 nM Minimal Yes (P-gp) M phase DM1 Tubulin ~0.1 nM Minimal (NCL ADC) Partial M phase DM4 Tubulin ~0.1 nM Moderate Partial M phase Calicheamicin DNA (DSB) ~10 pM Minimal Minimal Independent SG3199 (PBD) DNA crosslink ~pM Moderate Minimal Independent SN-38 Topo I ~1 nM Moderate Partial S phase DXd Topo I ~0.1 nM Strong Low S phase Alpha-amanitin RNA Pol II ~1 nM Moderate No Independent
Key Papers
Doronina et al. (2006) Bioconjug Chem — MMAE/MMAF comparison
Widdison et al. (2006) J Med Chem — DM1/DM4 maytansinoid design
Ogitani et al. (2016) Clin Cancer Res — DXd mechanism and bystander characterization
Jeffrey et al. (2010) Bioconjug Chem — PBD dimer ADC design
Modi et al. (2020) N Engl J Med — DESTINY-Breast01 T-DXd clinical validation