Destroying
cancerous cells
The role of diverse toxins in ADC development
Antibody drug conjugates (ADC) pair a monoclonal antibody with a cytotoxic payload to directly attack cancerous cells. The antibody drives tissue selectivity to enhance safety of an ADC, while the cytotoxic payload drives efficacy. A variety of cytotoxic payloads have been utilised in ADCs to date, and selecting an appropriate toxin is imperative to delivering an effective and successful ADC.
Cytotoxin
Payloads incorporated into ADCs must be highly cytotoxic to overcome a number of limitations of this therapeutic modality, including limited number of cell receptors and their saturation, and limited internalisation and accumulation in the cell. As a result, early ADC research and development has a focus on discovery and incorporation of increasingly cytotoxic payloads.
The following diagram shows the relative cytotoxicity of common ADC payloads.
Selecting cytotoxic payloads:
Toxin selection is a critical choice in ADC development, as the payload determines biochemical efficacy. To some extent all toxins have undergone research and development of their associated linkers with a view to enhancing beneficial properties, such as potency, tolerability and bystander effect, whilst minimising systemic toxicity. In many cases linkers used reflect a balance between these two aspects.
Select a toxin to learn more, or scroll on to read about seven of the most common cytotoxic payloads.
Maytansinoids
Maytansinoids are macrocyclic compounds which inhibit the assembly of microtubules by binding to tubulin. They prevent cell division, triggering apoptosis, ultimately leading to cell death.
Auristatins
Auristatins are peptidic compounds with antimitotic activity that induce depletion of cell microtubules and trigger the apoptotic process.
Tubulysins
Tubulysins are another peptidic tubulin interacting class which inhibits tubulin polymerisation and microtubule formation. Several derivatives retain activity in drug resistant cells.
Calicheamicins
Calicheamicins are a family of enediyne antitumour antibiotics that cleave DNA in a site-specific, double-stranded manner.
Anthramycins
Anthramycins are PBD antitumour antibiotics which inhibit DNA processing. Their high potency and cell cycle independent activity makes them ideal for treating tumours with low antigen expression and division rates.
Camptothecins
Camptothecins are pentacyclic alkaloids which inhibit DNA and RNA synthesis and cause DNA damage which eventually results in cell death. Unconjugated forms have successfully been developed.
Amatoxins
Amatoxins are a family of bicyclic peptidic compounds which inhibit RNA polymerase II resulting in reduced mRNA transcription, reduction of protein synthesis and cell death.
Maytansinoids
Target
Microtubule
History
First isolated in 1972 from the bark of the African shrub Maytenus Ovatus, maytansine was found to hold extremely potent cytotoxicity both in vitro and in vivo. Despite a lack of success in clinical trials on its own, its high potency, stability, and solubility in aqueous solutions made it attractive for utilisation in ADCs.
MOA
Maytansinoids work by preventing microtubule assembly. To do so, they bind to subunits of tubulin and interfere with microtubule polymerisation, leading to mitotic arrest and preventing cell division.
Structure and analogues
The structure of the payload has remained mostly unchanged since initial isolation, with minor modifications to add a thiol terminating extension, useful for generating conjugatable forms. In DM1 the thiol terminates a short ethyl chain while in DM4 the thiol terminates a longer propyl chain with two methyl groups adjacent to the thiol. DM1 is typically conjugated via a thioether while DM4 is typically conjugated via a disulfide linkage; the hindered thiol of DM4 is essential for conferring plasma stability to the disulfide. The thioether linked DM1 releases a charged derivative which has no bystander effect whereas the disulfide linked DM4 releases a thiol variant which can be S-methylated to form a cell permeable metabolite with bystander properties. Other modifications to DMx linkers include incorporation of charged groups, such as sulphate, to enhance ADC solubility.
Diagrams
Summary of structure-activity relationships (SARs) for maytansine
Maytansine (C35H48ClN3O10S) is made up of a 19-member ansamacrolide attached to a chlorinated benzene ring chromophore. Most SAR studies focus on correlations between structure and cytotoxic activities, rather than tubulin-binding properties.
Structures of maytansine, DM1, and DM4 ADC derivatives
DM1 and DM4 are the two main derivatives of maytansine, which differ in chain length and steric hindrance adjacent to the terminal thiol added to enable conjugation. Immunogen performed extensive studies comparing thioether and disulfide linked DMx ADCs and demonstrated that relative intrinsic conjugate potency in vitro and conjugate half-life in plasma does not directly predict efficacy in vivo. Processing of the DMx following release in the cell into more active metabolites with bystander activity is one additional factor contributing to the activity of these conjugates in vivo. These studies highlight the complexity of ADC development and optimisation. To date only a stable DM1 thioether derivative has been approved.
Auristatins
Target
Microtubule
History
Dolastatin 10 was originally isolated from the sea hare Dolabella Auricularia in 1987 and at the time was found to be the most potent antiproliferative compound known. It was found to display anticancer activity against a variety of indications, including lymphomas, leukaemia, and solid tumours. Because of its low therapeutic index and significant toxicity, however, it was dropped in phase II trials. Auristatins are synthetic, water soluble analogues of Dolastin 10 that have been used in cancer treatment for more than four decades.
MOA
Auristatins belong to a class of tubulin binding agents, and they have diverse chemical structures. Auristatins inhibit tubulin-dependent GTP binding to interfere with microtubule dynamics, causing mitotic cell cycle arrest and apoptosis.
Structure and analogues
Dolastin 10 has a pentapeptide structure, containing four amino acids which are unique to marine organisms. Synthetic auristatin derivatives have since been successfully exploited in ADCs. The two most common derivatives, MMAE and MMAF, are N-terminally modified with a mono methyl valine and C-terminus modifications of Ephedrine (E) and Phenylalanine (F) respectively, conferring differing solubility, hydrophobicity and bystander properties.
Diagrams
Summary of SAR for Dolastatin 10
Dolastatin 10 is a linear pentamer composed of four amino acids unique to marine organisms. Several modifications can be made to enhance cytotoxic activity.
Synthetic derivatives of Dolastatin 10
This figure shows synthetic derivatives of Dolastatin 10 which have been used in ADCs. All auristatin-based ADCs that have reached clinical trials to date are based on MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), or PF-06380101. Payload modifications contributing to its potency and selectivity are highlighted in teal, with purple indicating no change compared to Dolastatin 10.
Tubulysins
Target
Microtubules
History
Discovered in 2000 by Hofle and co-workers during a routine screen for novel secondary metabolites from myxobacteria, tubulysins display functional similarity to dolastatins and belong to the class of the most powerful mitotic inhibitors. They have been conjugated to antibodies, polymers, and small ligands.
MOA
Tubulysins bind to the tubulin’s vinca domain to cause depolymerisation. Depletion of microtubules causes cell cycle arrest and, ultimately, cell apoptosis.
Structure and analogues
There are approximately 14 reported isoforms of tubulysin to date, and naturally occurring tubulysins comprise a special, N,O-acetal moiety and either a tubutyrosine (Tut) or tubuphenylalanine (Tup) at the C-termini essential for their biological function. Because of their modular structure, tubulysins allow a variety of C-terminus modifications that do not affect activity.
Diagrams
Summary of SAR features for tubulysins
The amine group of the Mep residue at the N-terminus is essential for activity, the (R)-configuration of the O-acetate is preferred and affects tubulin binding kinetics. N,O-acetal groups can be eliminated without affecting cellular activity, and introducing a hydrophobic group improves bioactivity. The carboxylic acid group and the 3R configuration in the fragment D are essential for activity.
While tubulysins have shown effectiveness against numerous cancer cells that display rapid division rates, no tubulysin-based ADCs have been commercially approved to date. Their potent antiproliferative activity and resistance to the multidrug resistance (MDR) cell mechanisms have made them attractive in ADC development.
While tubulysins have shown effectiveness against numerous cancer cells that display rapid division rates, no tubulysin-based ADCs have been commercially approved to date. Their potent antiproliferative activity and resistance to the multidrug resistance (MDR) cell mechanisms have made them attractive in ADC development.
Calicheamicins
Target
DNA
History
Calicheamicins are produced by the bacterium Micromonospora echinospora ssp. calichensis, which was isolated in Waco, Texas in 1987. Too toxic when used alone, calicheamicin was found to be one of the most structurally complex natural products.
MOA
Calicheamicins induce double-stranded DNA damage. Calicheamicin diffuses into the cell nucleus and binds via oligosaccharide moiety to the minor groove of DNA. Next it undergoes re-arrangement to a highly reactive diradical moiety which attacks and destroys the DNA, causing single or double strand cleavage and cell apoptosis.
Structure and analogues
Calicheamicin γ1 is the most distinguished member of this class of compounds. Calicheamicins with iodine, which is obtained by treating a fermentation broth with sodium iodide, have shown better yields than those containing bromine. Calcheamicin itself was found to be too toxic for use in ADCs and a less potent N-acetyl-γ-calcheamicin was designed that contained modified amino sugar residues and a disulfide group replacing the trisulfide moiety. This design allowed for easier synthesis and a more stable molecule in circulation.
Diagrams
Summary of SARs
for calicheamicin
Calicheamicin (C55H74IN3O21S4). Essential groups and / or modifications which reduce activity are shown in green, and those that are acceptable or improve cytotoxicity are highlighted in blue.
Structure of N-acetyl-y-calicheamicin
N-acetyl-y-calicheamicin is less active in terms of DNA binding compared to the parent compound, but its potency, efficacy and superior therapeutic window makes it an attractive candidate in ADC technology.
Anthramycins
Target
DNA
History
Anthramycin was originally isolated from subtropical soils in the 1950s by M.D. Tendler. Leimgruber completed the total synthesis in 1965 and named the compound ‘anthramycin.’ The most well-known PBD dimer, a derivative of anthramycin, is SJG-136, which was synthesised in the 1990s and utilised in clinical trials for several types of cancer.1
MOA
Anthramycin and its derivative pyrrolobenzodiazepines (PBDs) bind covalently to guanine rich sequences in the minor groove of double-stranded DNA to block replication. They stall the DNA replication fork and block cell division, leading to cell death.
Structure and analogues
PBDs have two subfamilies: naturally occurring PBD monomers and synthetic PBD dimers. Relationship studies between the PBD structure and its biological activity have enabled more potent PBD dimers to be developed.
Diagrams
PBD monomer structure and its essential SARs
PBD monomer (C14H14N2O3). Anthramycin functional groups include a substituted aromatic A-ring, a diazepine B-ring and a pyrrolidine C-ring, with an S-chiral center.
Summary of SARs for PBD dimers
Several modifications can be made to PBD dimers to enhance potency and activity.
Dimers with C8/C8’ linkers
Only PBD dimers with C8/C8’ linkers can be arranged in the correct orientation to alkylate guanine in DNA. IGNs belong to a family of benzodiazepines reported to be more potent than SJG-136 due to their modification (changing the diimine form of the IGNs to monoimine). One imine moiety alkylates only single strand of DNA but have similar potency of diamine IGNs without o-target toxicity.
Stereoisomerism
Stereoisomerism is crucial for PBD activity. Isoquinolidinobenzodiazepine (IQB) dimers belong to a new class of PBD dimer family and display even higher potency than IGNs. The substitution of the pyrrolidine ring by an indoline ring has been shown to increase payload activity.
Camptothecins
Target
Topoisomerase I inhibitor
History
Used for centuries in traditional Chinese medicine, camptothecin’s antitumour properties were rediscovered by western medicine in 1958. Three water soluble camptothecin derivatives have been approved in the last 25 years to be used as standalone treatments for a variety of cancers.
MOA
Camptothecins inhibit the DNA replication process through interaction with Topoisomerases I, a group of enzymes that cut, relax, and reanneal one or two-stranded DNA breaks. In turn, this leads to inhibition of DNA replication and transcription, chromatin condensation, nuclear fragmentation, cell cycle arrest, and, finally, cell apoptosis.
Structure and analogues
Camptothecin has a planar pentacyclic ring structure, thought to be a critical factor in topoisomerase inhibition. Since natural camptothecin displays low solubility, clinical analogues aim to improve solubility as well as stability of the lactone ring. Three FDA-approved, water soluble analogues have been developed: topotecan, irinotecan and belotecan.
Diagrams
Structure of camptothecin and summary of its SARs
Camptothecin (C20H16N2O4). Essential groups, ie. Modifications that reduce activity, are shown in teal, and those that improve cytotoxicty are shown in in blue.
Exatecan
A unique semi-synthetic derivative, exatecan has a unique hexacyclic structure with an extra ring between C7 and C9, increasing solubility. It is more potent than other analogues, and it lacks esterase-dependent activation step.
SN-38
SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite of irinotecan, with 1000-fold greater activity thanks to C7 alkyl and C10-, C20- hydroxyl group substitutions.
Amatoxins
Target
RNA-polymerase inhibitor
History
α-Amantin was characterised in the 1950s by Wieland and was found to be present in several poisonous mushroom species, such as the death cap (Amanita phalloides) and destroying angel (Amanita bisporigera) α-Amantin is the deadliest of all amatoxins, accounting for the majority of fatal mushroom poisonings. Because of delayed onset symptoms, amatoxin positioning is difficult to diagnose.
MOA
As the most potent inhibitor of mRNA transcription, α-amantin binds covalently to RNA polymerase II, a multiprotein complex responsible for transcribing DNA into mRNA.
Structure and analogues
At least 10 amatoxin analogues can be distinguished, with α-amantin being the major form. Compared to existing payloads, α-amantin delivers superior properties such as hydrophilicity that facilitate conjugation in aqueous buffers and prevent aggregation of the ADC.
Diagrams
α-amantin structure
α-amantin, C39H54N10O14S, is the major form of amatoxin. Functional groups that may be used as linker attachment sites are circled, and potency-reducing modifications are noted.
HDP 30.2115
A synthetic α-amantin, HDP 30.2115 contains tryptophan and a thioether bond at the asparagine. This derivative is used in HDP-101, an ADC candidate currently in clinical-stage development.
While no amatoxin-based ADCs have received commercial approval, HDP-101 is an emerging candidate currently in clinical-stage development. Preclinical data showed strong in vitro antitumour activity, leading to complete tumour remission in mouse models for multiple myeloma at very low doses.
Optimising toxins to maximise ADC success
As of 2021, 11 ADCs have received commercial approval, with many more in various stages of clinical trials. To date, more than 80 ADCs harnessing various cytotoxic payloads for a range of cancer indications have been involved in over 600 clinical trials worldwide.2
While commercially approved ADCs to date use 6 different classes of toxins, many more have been and continue to be explored in ADC development. Ongoing research and application of various cytotoxic payloads in will support the development of a variety of ADCs for various cancers and other indications, with research now expanding to examine ADCs in infectious, inflammatory, and autoimmune diseases.
ADCs today
11 ADCs
have received commercial approval
Over 80 ADCs
have been involved in clinical trials.
600+ ADC
clinical trials have taken place worldwide.
Tailored ADC development, supported by rich expertise
At Sterling, our dedicated bioconjugation team has more than 35 years of expertise in custom, quality-focused ADC discovery and development. Our team’s experience working with a variety of target molecules, linkers, and toxins, paired with our focus on true scientific partnership, enables us to support our customers in developing tailored ADCs that meet their unique requirements.
- Mantaj, J.; Jackson, P.; Rahaman, K.; Thurston, D. From Anthramycin to Pyrrolobenzodiazepine (PBD)‐Containing Antibody–Drug Conjugates (ADCs). Angew Chem Int Ed Engl [Online], Jan 9, 2017, 462-488. National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215561/ (accessed June 8, 2021).
- Zhao, P.; Zhang, Y. et. al. Recent advances of antibody drug conjugates for clinical applications. Acta Pharmaceutical Sinica B [Online], September 2020, 1589-1600. ScienceDirect. https://www.sciencedirect.com/science/article/pii/S2211383520305554 (accessed June 8, 2021).