History of ADC Payloads and Its Pharmacokinetic Profiles

Chemotherapy drugs are an important means of tumor treatment. At present, chemotherapy drugs based on different mechanisms have been developed, which can act on the initial raw materials such as purine and pyrimidine required for upstream nucleotide synthesis, as well as deoxynucleotides required for DNA replication or ribonucleotides required for RNA transcription, or DNA, RNA, and proteins that act directly on the “central dogma.” As an important component of the cytoskeleton, tubulin participates in many cellular processes that are critical to cell function, such as cell migration and mitosis. As a classic anticancer drug, paclitaxel acts as a tubulin stabilizer, while vinblastine acts as a microtubule destabilizer.

In order to enhance the targeting of chemotherapy drugs, since the late 1950s, researchers have successively developed conjugates composed of mouse antibodies and chemotherapy drugs such as doxorubicin, vinca alkaloids and methotrexate. Conducting preclinical research and clinical research has greatly promoted the research and development of ADC drugs. It can be said that ADC was born out of chemotherapy drugs. However, the first-generation ADCs were not successful, mainly because the targeting of the first-generation ADCs was poor, and the activity of the chemotherapy drugs themselves was not strong enough, resulting in unsatisfactory curative effect but not low toxicity. Some highly toxic compounds cannot be used as drugs alone because they are too toxic, but they may be used as payloads of ADC drugs. With the gradual clinical success of ADC drugs, and the combination of ADC drugs and immune checkpoint inhibitors is likely to replace the combination of chemotherapy drugs and immune checkpoint inhibitors, ADC drugs will surely have a wider space for development. The ideal ADC payload properties include:

▪ Unique mechanism of action, which is different from the mechanism of commonly used chemotherapeutic drugs, can effectively avoid cross-resistance;

▪ The lethality to tumor cells is strong enough to reach the pM level;

▪ There is a suitable site for linker ligation;

▪ Has certain water solubility, which can promote the coupling of ADC;

▪ Rapid elimination in the blood circulation system, reducing systemic toxicity;

▪ Substrates of non-efflux transporters to avoid drug resistance;

▪ It is best to have a bystander effect and kill heterogeneous tumors.

Highly Effective Cytotoxic Drug

The payloads of currently approved ADC drugs are highly toxic, mainly including MMAE/MMAF, calicheamicin, DM1/DM4, SN38/Dxd, etc. The toxicity of these payloads is 1-2 orders of magnitude higher than that of chemotherapy drugs , and some toxicity even reached the pM level. The mechanisms of action of currently developed payloads are largely limited to DNA alkylating agents, DNA topoisomerase inhibitors, tubulin disruptors, and RNA polymerase III inhibitors. Table 1 summarizes the mechanism of action of the ADC payload, whether there is a bystander effect, and the associated toxicity caused by the payload, etc. However, there are not many highly effective cytotoxic drugs. Therefore, there is an urgent need to develop cytotoxic drugs with high toxicity and different mechanisms of action.


Ducamycin is a DNA alkylating agent, which can act on the whole cycle of tumor cells, and its in vitro activity reaches pmol level. Ducamycin has good membrane permeability, has a bystander effect, and maintains activity against cell lines containing multidrug-resistant efflux.

SYD985 is a HER2-targeting ADC developed by Byondis. Trastuzumab is linked to ducamycin through a cleavable linker, with a DAR value of 2.8. After SYD985 is cleaved by cathepsin B, the free phenol promotes intramolecular rearrangement into an electrophilic cyclopropyl form, and cyclopropane is easy to covalently bind to DNA, resulting in DNA alkylation. The main safety concerns of SYD985 include ocular toxicity and interstitial pneumonia (ILD).


Osteosarcoma is the most common malignant bone tumor in children and adolescents. About 40-50% of patients may relapse or develop distant metastasis after surgery, resulting in a 5-year survival rate of less than 30%. Anthracyclines such as doxorubicin are currently one of the first-line chemotherapeutic drugs for osteosarcoma, which can insert into the DNA double helix, prevent the separation of the double strands, and affect DNA replication and RNA synthesis. Due to the insufficient cytotoxicity of doxorubicin as an ADC payload (Fig. 2), another anthracycline PNU-159682 with 100-fold higher cytotoxicity than doxorubicin was developed. PNU-159682 is a hepatic metabolite of Nemorubicin that inhibits DNA topoisomerase II with three orders of magnitude higher potency than Nemorubicin with IC50 of 20-100 pM, and PNU-159682 is not a substrate of efflux transporters.


The amanitin family consists of nine structurally similar toxins, derived from the world’s most toxic mushroom Amanita, among which α-amanitin and β-amanitin are the two most important toxins, which are bicyclic peptides composed of 8 amino acids. Ingestion of amanitas leads to vomiting, seizures, and severe liver damage (amanitin is a substrate of the liver transporter OATP1B3), which is responsible for 90% of mushroom-related deaths. Amanitin is a highly efficient RNA polymerase III inhibitor with a cytotoxicity range of pM, which can efficiently bind to eukaryotic RNA polymerase II to reduce the efficiency of RNA transcription and protein synthesis by more than 1000 times. Therefore, amanitin can not only kill tumor cells in the dividing stage, but also kill cells in the dormant stage, including tumor stem cells. Due to too high toxicity, it cannot be further developed as an anticancer agent, but it presents many advantages as a potential ADC payload, such as high toxicity, hydrophilicity, non-P-glycoprotein (P-gp) substrate, etc.


Tubulysin-like natural products are toxic tetrapeptides isolated from myxobacteria species that inhibit microtubule polymerization. The cytotoxicity of this kind of natural products is more than 10-1000 times that of vinblastine and paclitaxel (IC50 is in the picomolar range), and cells are arrested in G2/M phase by inhibiting microtubule polymerization. And Tubulysin is not a substrate of multidrug resistance efflux transporters. Drug developers have been working on the structure-activity relationship (SAR) of Tubulysin-like natural products, hoping to use this type of molecule as a payload for the development of new ADCs.

Source: https://www.bocsci.com/blog/history-of-adc-payloads-and-its-pharmacokinetic-profiles/