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
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).
- PNU-159682
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.
- Amanitin
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.
- Tubulysins
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.
AstraZeneca/MedImmune has developed a bispecific ADC, MEDI4276, targeting two different epitopes of HER2, the bispecificity may increase internalization, thereby increasing payload release and enhancing killing of cancer cells. MEDI4276 is connected by a bispecific antibody and Tubulysin derivative AZ13599185 through a non-cleavable linker, with a DAR value of 4. MEDI4276 has shown good antitumor activity in preclinical studies, and the main toxicity in clinical studies is liver toxicity.
- Eribulin
Eribulin is a synthetic analogue of the macrocyclic Halichondrin b isolated from sponges. Eribulin selectively binds to the (+) end of β-tubulin, inhibits microtubule elongation, but has little effect on microtubule depolymerization. Therefore, Eribulin is still effective for patients who are resistant to paclitaxel. In November 2010, Eribulin was approved by the US FDA for the treatment of metastatic breast cancer. The antibody-drug conjugated ADC with Eribulin as cytotoxin has a strong bystander effect, so it has better clinical effect, and Eribulin is less sensitive to P-glycoprotein (P-gp).
In Vitro and In Vivo Pharmacokinetic Studies of Payload
- Plasma Protein Binding Studies
After ADC administration, the concentration of payload in plasma is generally low, and the investigation of plasma protein binding should examine the lower concentration range of payload. However, due to the limitation of detection sensitivity, when conventional mass spectrometry methods cannot detect, radioactively labeled payloads can be used to investigate the plasma protein binding rate.
- Tissue Distribution and Excretion/Material Balance
Tissue distribution and excretion studies using conventional mass spectrometry methods are challenging due to detection sensitivity limitations. Using non-radioactively labeled payloads for research, it is difficult to ensure a high excretion recovery rate, clarify the excretion route of the payload, and obtain important information on whether the highly toxic payload can be successfully excreted from the body. Therefore, for payloads, a radiolabeled approach is often recommended to track the ADME signature of small molecule compounds in vivo. For tissue distribution studies, quantitative whole-body autoradiography (QWBA) is generally recommended. QWBA can display the distribution of payload in various tissues in more detail, especially the skin, eyes and other tissues that are difficult to distinguish by tissue harvesting methods.
- In Vitro and In Vivo Metabolism Studies
From the perspective of metabolic stability, the ideal payload needs to be rapidly metabolized once it enters the circulatory system, thereby reducing systemic exposure and systemic toxicity. It is necessary to understand whether there are species differences in payload metabolism. The selection of toxicity-related animal species needs to refer to the characteristics of payload metabolism, and select species related to humans for toxicological investigations. In addition, it is also recommended to use plasma collected in toxicokinetics (TK) experiments to carry out payload-related metabolite identification studies. And compared with the identification results of payload-related metabolites in plasma after ADC administration in humans, to evaluate whether the exposure of payload-related metabolites in toxicological species can cover human species.
- Effects on Metabolic Enzymes
This evaluation includes the presence or absence of inhibition or induction of drug-metabolizing enzymes by the payload and the identification of drug-metabolizing enzyme subtypes involved in payload metabolism. Theoretically, the payload has a low blood concentration in the body and has a low risk of acting as an inhibitor or inducer of metabolic enzymes. Also due to the low blood drug concentration in the payload, if the payload is known to be a substrate of a metabolic enzyme, inhibition of the enzyme may significantly increase the blood drug concentration in the payload, thereby increasing the toxic response. Therefore, phenotyping of payload-metabolizing enzymes is crucial in the preclinical research phase.
- Effects on Transporters
This evaluation includes whether the payload is a transporter substrate or a transporter inhibitor. Due to the generally low concentration of the payload in the body, theoretically, the risk of the payload acting as a transporter inhibitor is low. Transporter uptake or efflux affects the distribution and excretion of drugs in the body. If the payload is an uptake or efflux transporter substrate, it can affect the distribution of the payload in tissues with high expression of certain transporters, increasing the risk of toxicity to specific tissues. Tumor tissues generally overexpress efflux transport-related proteins such as Pgp, BCRP or MRPs. If the payload is a substrate of Pgp, BCRP or MRPs, the effect of the efflux transporter will reduce the accumulation of the payload in the tumor tissue, thereby affecting the efficacy and producing drug resistance.
- PK/PD Research
Although the antibody portion of the ADC may interact with immune effector cells, including CDC (complement-dependent cytotoxicity), ADCC (antibody-dependent cellular cytotoxicity) and ADCP (antibody-dependent cellular phagocytosis) effects, the pharmacodynamics of the ADC mainly come from the payload. Zhang et al. compared the drug efficacy of different ADCs with PBD as payload on tumor mice. The antitumor effect was directly related to the exposure of the payload in the tumor. Meanwhile, the antitumor effect levels off over time due to saturation of the target (DNA) response to the payload. The ADC with MMAE as the payload also has a similar phenomenon in the drug effect model of tumor-bearing mice. This study suggests that the antitumor efficacy of ADC is not related to the systemic exposure of ADC, nor to the exposure of the payload in plasma, but directly related to the exposure of the payload in the tumor. In view of this, it is necessary to carry out PK studies of tumor tissues in model animals to evaluate the PK/PD of ADC drugs.
References
- Goldenberg, D.M. et al. Sacituzumab govitecan, a novel, third-generation, antibody-drug conjugate (ADC) for cancer therapy. Expert Opin Biol Ther. 2020, 20(8): 871-885.
- Fuentes-Antrás, J. et al. Antibody-drug conjugates: in search of partners of choice. Trends Cancer. 2023, 9(4): 339-354.
- Li, J.Y. et al. A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy. Cancer Cell. 2016, 29(1): 117-29.
