Among the first-generation ADC drugs, traditional chemotherapy drugs such as methotrexate, vinblastine, and doxorubicin were used as cytotoxic payloads. However, due to insufficient cytotoxicity and selectivity against tumor cells, the first-generation ADC had a low target cell uptake rate, and its efficacy was even inferior to the payload itself, resulting in the failure of its clinical development. Subsequently, new highly cytotoxic compounds that are 100 to 1,000 times more potent than traditional chemotherapy drugs used in first-generation ADCs have attracted widespread attention from researchers. These payloads often cause strong side effects when used as a single drug that kills tumors. For example, the tubulin inhibitor shows strong anti-proliferative activity against tumor cells. However, its neurotoxicity and gastrointestinal effects are also serious. Therefore, tubulin is not approved as a single agent for cancer treatment. Most second-generation ADCs use more potent tubulin inhibitors as payloads. While tubulin inhibitors are very effective against dividing tumor cells, they are much less effective against quiescent cancer cells. To overcome this limitation, DNA-damaging agents capable of targeting the entire cell cycle serve as cytotoxic payloads for most third-generation ADCs. DNA damaging agents can destroy the DNA structure through double-strand breaks, alkylation, chimerization and cross-linking, thereby killing tumor cells. Representative DNA damage payloads include enedynes, topoisomerase I inhibitors, and pyrrolobenzodiazepines (PBDs). Currently, a total of 15 ADC drugs have been approved globally, and a large number of ADC drugs are undergoing clinical trials (Fig. 1).
Classification of ADC Payloads
The payload is the component that exerts the tumor-killing effect. After the ADC drug enters the cell, the payload is the main force that ultimately causes the death of the target cell. Therefore, the toxicity and physical and chemical properties of the payload will directly affect the drug’s ability to kill tumors, thereby affecting the efficacy. The payload used for conjugation must have a clear mechanism of action, a small molecular weight, high cytotoxicity, and still retain anti-tumor activity after being chemically coupled to the antibody. In addition, other properties of the ADC payload such as immunogenicity, stability during preparation and circulation, water solubility, and modifiability are also important.
- Microtubule-targeting Payloads
Microtubules are an important component of the eukaryotic cytoskeleton. Microtubules play an important role in maintaining cell morphology, signal transduction, organelle transport, cell movement, cell division, mitosis and other cellular functions, and are important targets for tumor treatment. Microtubules are composed of tubulin. By binding to tubulin, tubulin inhibitors inhibit cells from entering the G2/M phase of the cell cycle, interfere with the dynamic combination of cells, and ultimately lead to cell apoptosis. Because tubulin inhibitors disrupt the mitotic spindle and exert antimitotic effects, they are more toxic to rapidly dividing tumor cells than to slow-growing normal cells. Therefore, tubulin inhibitors that interfere with tumor cell mitosis have become one of the directions for tumor drug development. This type of inhibitor mainly inhibits the polymerization of tubulin or promotes the polymerization of tubulin. Among them, tubulin polymerization-promoting agents act on the β-subunit of α-β tubulin dimer, making microtubule growth unregulated, such as MMAE and MMAF. Agents that inhibit tubulin polymerization block the polymerization of tubulin dimers by inhibiting the formation of mature microtubules, such as DM1 and DM4.
Maytansinoids: Maytansine originally isolated from the bark of an African shrub, is an antibiotic that binds to tubulin and inhibits microtubule assembly. In in vitro cell activity tests, its IC50 is in the picomolar range, indicating that it has a strong ability to inhibit tumor cell proliferation. Good stability and solubility were also shown in other experiments.
Auristatin: Dolastatin10 was isolated from black fungus in 1987. It has strong anti-proliferative activity against a variety of cancer cells and can strongly inhibit microtubule assembly, leading to cell cycle arrest and apoptosis, becoming a potential of anticancer drugs. Its water-soluble synthetic analog is called Auristatin. Among them, the most widely used ones are MMAE and MMAF. Both MMAE and MMAF contain a modifiable functional group. MMAE is composed of four amino acids: monomethylvaline (MeVal), valine (Val), dolaisoleuine (Dil) and dolaproine (Dap), as well as norephedrine. In MMAF, the C-terminus of monomethylvaline is replaced by phenylalanine, and its cellular activity is significantly reduced. The binding affinity of MMAF to free tubulin is nearly 5-fold higher than that of MMAE, mainly due to the critical Arg278 residue on the β1 tubulin subunit, which is exposed to MMAF through ordered water molecules.
Eribulin: It is a natural product of polyether macrolides, halichondrin B, which has been shown to have good anti-proliferative activity. By modifying its structure, a new non-taxane total synthetic analogue of tubulin methanesulfonate, a microtubule kinetic inhibitor with anti-mitotic effect, was approved in 2010 for the treatment of patients with locally advanced and metastatic breast cancer (MBC).
Tubulysins: Tubulysins is a natural antimitotic peptide isolated from slime mold culture medium by Hofle et al. It is composed of N-methyl-D-piperonic acid (Mep), lysosine (L- A linear tetrapeptide composed of Ile), toubvaline (Tuv), and tubulin alanine (Tup) or microtubule hemolysin Tut (Tut). It can inhibit tubulin polymerization and induce apoptosis. It has strong anti-proliferative activity against multidrug-resistant KB-V1 cells (half inhibitory concentration Z 0.08 nmol/L). It has a good application prospect in the development of anti-cancer drugs.
Cryptophycins: Cryptophycins are a natural macrocyclic polypeptide produced by cyanobacteria and have a 16-amino acid composition. It contains two hydroxy acids (A and D units) and two amino acids (B and C units). Cryptophycins irreversibly inhibit β-tubulin polymerization during mitosis, causing cell cycle arrest in the G2/M phase and activating the apoptotic pathway, resulting in picomolar antiproliferative capabilities in vitro.
EG5 inhibitor: Kinesin (KSP/EG5/KIF11) is an ATP-dependent protein that participates in the separation of centrosomes and the production of spindles in the G2/M phase of the cell cycle, and plays an important role in mitosis. High expression of EG5 in hematological tumors, such as AML and diffuse large B-cell lymphoma (DLBCL), and solid tumors, such as breast, bladder, and pancreatic cancer, is associated with poor prognosis in these diseases. Some EG5 inhibitors, such as SB-715992 (Ispinesib) and ARRY-520 (Filanesib), are in clinical trials.
- DNA-targeting Payloads
Compared with tubulin inhibitors, DNA inhibitors can damage DNA through double-strand breaks, alkylation, chimerism, cross-linking, etc. It acts on the entire cell cycle, produces cytotoxic effects, and has a good therapeutic effect on solid tumors. In addition, DNA inhibitors have fewer targets than tubulin inhibitors and can exhibit better killing effects when the ADC carries the same amount of payload into the cell. Furthermore, ADCs with DNA inhibitors as payloads can target tumor cells with low antigen expression, which also explains why DNA inhibitors are chosen as payloads in many next-generation ADCs.
Enediyne: Enediyne is one of the most cytotoxic natural products discovered to date. By destroying single-stranded or double-stranded DNA in living cells, leading to cell death, it has extremely strong activity against tumor cells. Enediyne can be divided into two subfamilies: calcineurin-like Enediyne and anthraquinone-fused Enediyne. These highly toxic Enediyne are not suitable for direct use as anticancer drugs, and the most commonly used ADC payloads of natural Enediyne products are calcimycin gI 1 and anthraquinone-fused enedinomycin.
Topoisomerase I Inhibitors: DNA Topoisomerase I (topo I) is an enzyme that cuts DNA strands. The DNA relaxing activity of DNA topoisomerase I is indispensable for transcriptional repression and has become a popular target for ADCs. ADCs targeting topo I may work through immunotherapy. Lance Stewart et al. reported the x-ray crystal structure of TOPO-I, used in combination with the approved anti-tumor drug topotecan, covalently bound to double-stranded DNA. Covalent complexes of topotecan and TOPO I-DNA indicate that topotecan is a noncompetitive inhibitor that binds to the enzyme substrate complex by inserting a base between the two DNA strands at the enzyme-induced nick. The binding mode of topotecan is stacking interaction with DNA, hydrogen bonding contact with Asp-533, and water bridge contact with the active site between phosphotyrosine and Asn-722.
Pyrrolobenzodiazepine (PBD): PBD found in Streptomyces species are a class of natural products with antitumor activity. Its mode of action is that PBD selectively alkylates in small grooves in DNA, where the N2 of DNA guanine forms a covalent bond with the electrophilic N10/C11 imine of PBD. Sustained DNA damage is caused by cross-links between DNA strands, which arrests the cell cycle in the G2/M phase, leading to cell apoptosis, thus exhibiting a powerful cytotoxic effect. PBD dimers have the potential to be used as ADC payloads. For example, ROR1 targeting ADC CS5001 designed with PBD as payload shows strong selectivity in a variety of tumor cell lines expressing ROR1, and shows significant in vivo anti-tumor activity in blood and solid tumor xenograft mouse models. Clinical trials are currently underway.
Duocarmycins: Duocarmycin A is a strong DNA alkylating agent isolated from Streptomyces, including a DNA alkylating moiety and a binding moiety. The first discovered Duocarmycins member, CC-1065, binds to DNA microgrooves through its highly active propane ring and alkylates adenine at the N3 position, ultimately leading to cell death. Although CC-1065 is highly effective in vitro, it shows only moderate in vivo activity and irreversible hepatotoxicity in animal models. To improve the biological properties of these compounds, their use in ADCs may be a viable option.
Judging from the IC50 distribution of toxins, ADC cytotoxicity must be at least nM level. Among the currently more commonly used cytotoxins, maytansine is more toxic than Aplysia toxin, and the most toxic one is currently PBD among DNA damaging agents, which is one of the relatively effective payloads. PBD kills cancer cells by binding to and cross-linking to specific targets on the DNA of cancer cells. This could therefore prevent tumor cells from proliferating without deforming their DNA helices, potentially avoiding the development of drug resistance.
References
- Wang, Z. et al. Antibody-drug conjugates: Recent advances in payloads. Acta Pharmaceutica Sinica B. 2023, 13(10): 4025-4059.