Antibody-drug conjugates (ADCs) are highly precise new class of biopharmaceutical products that covalently linked a monoclonal antibody (mAb) (binds explicitly to a tumor-associated surface antigen) with a customized cytotoxic drug (kills cancer cells) and tied via a chemical linker (releases the drug). Due to its precise design, it brings about the target cell killing sparing the normal counterpart and free from the toxicities of conventional chemotherapy.
The payload is a crucial component of ADCs, as its activity and physicochemical properties directly influence the anti-tumor efficacy of the drug. Payloads for ADCs can be classified into several categories, including microtubule inhibitors, DNA-damaging agents, topoisomerase inhibitors and RNA polymerase II inhibitors, etc.
Currently, the variety of payloads available for marketed ADCs is limited. The most prominent clinical-stage payloads include MMAE, MMAF, DM1, DM4, SN-38, DXd, and PBD, etc. There is an urgent demand for the development of next-generation ADCs featuring novel payload mechanisms. This article will briefly introduce several newly developed non-traditional payloads, including eribulin, duocarmycin, PNU-159682, amanitin and tubulysins, etc.
Target | Mechanism of Action | Payload | Bystander Effect | Representative Drugs | |
DNA | DNA-damaging agents | Calicheamicin | Yes | Besponsa | |
Duocarmycins | Yes | SYD895 | |||
Pyrrolobenzodiazepine (PBD) | Yes | Zynlonta | |||
Topoisomerase I inhibitors (TOP1) | Camptothecin & SN38 | Yes | Trodelvy | ||
Dxd | Yes | Enhertu | |||
Exatecan & derivatives | Yes | AZD8205 | |||
Topoisomerase II inhibitors (TOP2) | PUN-159682 | N/A | SOT102 | ||
RNA | RNA polymerase II inhibition | Amanitin | No | HDP-101 | |
Microtubule | Microtubule inhibitor | Auristatins | MMAE | Yes | Padcev |
MMAF | No | Blenrep | |||
Maytansinoids | DM1 | Yes | Kadcyla | ||
DM4 | Yes | Elahere | |||
Tubulysins | N/A | MEDI4276 | |||
Halichondrin (Eribulin) | Yes | MORAb-202 |
Table 1. Overview of ADC payloads
Novel ADC Payloads
Eribulin
Eribulin is a synthetic analogue of marine natural product halichondrin B, a large polyether macrolide derived from a marine sponge called Halichondria okadaic. It primarily binds to the β-tubulin at microtubule plus ends, effectively blocking microtubule polymerization while having little to no effect on microtubule depolymerization or shortening. This mechanism allows eribulin to remain effective in patients who have developed resistance to paclitaxel.
In November 2010, the U.S. FDA approved eribulin for the treatment of metastatic breast cancer. As a cytotoxic component in ADCs, eribulin exhibits a significant "bystander effect," contributing to its favorable clinical outcomes, and it shows reduced sensitivity to P-glycoprotein (P-gp).
Farletuzumab Ecteribulin, also known as MORAb-202, is an ADC developed by Eisai that targets FRα. It comprises three components: the FRα monoclonal antibody farletuzumab, the microtubule inhibitor eribulin, and a cleavable linker, with a drug-to-antibody ratio (DAR) of 4, which enhances its bystander effect. Currently, it is undergoing clinical Phase II trials. At the ASCO 2022 conference, Eisai presented data showing an overall response rate (ORR) of 31.6% at a dose of 0.9 mg/kg and 50.0% at 1.2 mg/kg. The most commonly reported adverse reaction was interstitial lung disease (ILD), although it was primarily of low grade.
Figure 1. Structure of MORAb-202
Duocarmycins
Duocarmycins and analogues are DNA-alkylators highly cytotoxic to cancer cells. It is typically conjugated at the phenolic functional group and effectively binds to the minor groove of DNA, leading to N3 alkylation of adenine and resulting in the destruction of tumor cells. Duocarmycin has good membrane permeability, exhibits a bystander effect, and retains activity against cell lines with multidrug resistance mechanisms.
SYD985 (trastuzumab duocarmazine ), developed by Byondis, is a HER2-targeting ADC. It links trastuzumab to duocarmycin via a cleavable linker, achieving a DAR of 2.8. The antibody part of SYD985 binds to HER2 on the surface of the cancer cell and the ADC is then internalized. After proteolytic cleavage of the linker, the inactive cytotoxin is activated and DNA damage is induced, resulting in tumor cell death.
Figure 2. Structure of SYD985 [4]
On July 12, 2022, the FDA accepted the Biologics License Application (BLA) for SYD985 to treat HER2-positive metastatic breast cancer. However, on May 15, 2023, Byondis announced that the FDA did not approve SYD985 for market release, citing the need for additional information to support their decision. The primary safety concern associated with SYD985 is ocular toxicity, with 38.2% of patients experiencing keratitis or conjunctivitis. Additionally, there are reports of interstitial pneumonia, affecting 7.6% of patients, with two fatalities attributed to this condition. Notably, SYD985 also has a narrow therapeutic window.
PNU-159682
Anthracycline drugs, such as doxorubicin, are classic chemotherapy agents that insert into the DNA double helix, preventing strand separation and disrupting DNA replication and RNA synthesis. However, doxorubicin's toxicity is insufficient for use as an ADC payload, prompting the development of PNU-159682, an anthracycline with 100 times greater potency. PNU-159682 is a liver metabolite of nemorubicin and effectively inhibits DNA topoisomerase II, demonstrating an efficacy three orders of magnitude higher than that of nemorubicin, with an IC50 of 20–100 pM. Notably, PNU-159682 is not a substrate for efflux transport proteins, enhancing its therapeutic potential.
SOT102 is a CLDN18.2-targeting ADC developed using a proprietary monoclonal antibody conjugated in a sitespecific manner via a noncleavable amide/peptide linker to a derivative of the highly potent anthracycline PNU159682 payload in a DAR2 light chain format. In various patient-derived xenograft (PDX) models, SOT102 has demonstrated significant single-agent activity, achieving complete responses (CR) across all CLDN18.2-positive models, regardless of staining intensity.
Figure 3. Structure of SOT102
Amanitin
Amanitin, a toxin derived from Amanita phalloides (death cap), acts as a highly potent inhibitor of RNA polymerase II, effectively halting protein synthesis by significantly reducing RNA transcription, making it capable of killing both actively dividing and dormant cancer cells, including cancer stem cells. Although its extreme toxicity prevents direct use as a cancer treatment, its high potency, water-solubility, and lack of interaction with P-glycoprotein make it a promising candidate as a payload in ADCs for targeted cancer therapy.
HDP-101 is an ADC targeting BCMA developed by Heidelberg Pharma, consisting of a BCMA antibody, a VA-PABC linker, and an amanitin derivative known as HDP 30.2115. Due to its high toxicity, the DAR is set at 2, and a site-specific coupling method is employed to ensure uniformity and mitigate potential toxicity. In May 2021, HDP-101 entered early clinical trials for the treatment of multiple myeloma (NCT04879043). Recent findings indicate that HDP-101 acts as an immune activator, capable of inducing immunogenic cell death (ICD) and demonstrating synergy with immune checkpoint inhibitors, opening new possibilities for combination therapies in the clinic.
Figure 4. Structure of HDP-101
Tubulysins
Tubulysins are a group of naturally occurring compounds extracted from myxobacteria that are highly effective at inhibiting microtubule polymerization, demonstrating significantly greater cytotoxicity compared to standard chemotherapy drugs like vincristine and paclitaxel, often exhibiting IC50 values in the picomolar range, meaning they can kill cancer cells at extremely low concentrations; essentially making them very potent anti-cancer agents. Researchers have been focused on exploring the structure-activity relationships (SAR) of tubulysin derivatives, aiming to utilize these compounds as effective payloads in the development of new ADCs.
MEDI4276 targets two epitopes, ECD2 and ECD4, of the HER2 receptor. It employs a cleavable peptide linker to conjugate the microtubule inhibitor tubulysin (AZ13599185) to the antibody, achieving a DAR of approximately 4. Phase I clinical trials have been conducted to evaluate its safety and preliminary efficacy in patients with gastric and breast cancer, with doses ranging from 0.05 to 0.9 mg/kg administered every three weeks.
Figure 5. Structure of MEDI4276
Results indicated that doses of 0.4, 0.6, and 0.9 mg/kg resulted in dose-limiting toxicities (DLTs). Notably, one breast cancer patient achieved a complete response (CR) at 0.5 mg/kg, while two patients achieved partial responses (PR) at doses of 0.6 and 0.75 mg/kg. The maximum tolerated dose (MTD) for MEDI4276 was found to be less than 0.3 mg/kg, with effective doses exceeding this threshold and indicating a very narrow safety window. Since the initiation of Phase I trials in 2015 and the data disclosure in 2021, there has been no further progress in its development.
Conclusion
ADCs offer considerable promise in cancer treatment, but current payloads face several challenges. These include limited penetration in solid tumors, variable efficacy against certain cancers, complex pharmacokinetics, and a propensity for resistance.
To overcome these limitations, there is a pressing need to develop novel, effective payloads with fewer side effects. Strategies such as screening natural product libraries, employing chemical synthesis, and directly modifying existing natural products can help optimize current payloads. By enhancing the therapeutic potential of ADCs, we can better address the complexities of cancer treatment.
Some ADCs, like MORAb-202 and SYD985, use PEG in their linkers. PEG enhances the loading capacity of ADCs by creating a protective shield around the payload, which improves solubility and stability. This approach reduces aggregation and immunogenicity, leading to better pharmacokinetics and decreased toxicity. Biopharma PEG offers a variety of PEG linkers to facilitate antibody-drug conjugate (ADC) development projects. All PEG linkers are of >95% purity and they are the basic building blocks for a successful ADC.
References:
[1] Goldenberg DM, Sharkey RM. Sacituzumab govitecan, a novel, third-generation, antibody-drug conjugate (ADC) for cancer therapy. Expert Opin Biol Ther. 2020 Aug;20(8):871-885. doi: 10.1080/14712598.2020.1757067. Epub 2020 May 12. PMID: 32301634.
[2] Fuentes-Antrás J, Genta S, Vijenthira A, Siu LL. Antibody-drug conjugates: in search of partners of choice. Trends Cancer. 2023 Apr;9(4):339-354. doi: 10.1016/j.trecan.2023.01.003. Epub 2023 Feb 4. PMID: 36746689.
[3] Nakada T, Sugihara K, Jikoh T, Abe Y, Agatsuma T. The Latest Research and Development into the Antibody-Drug Conjugate, [fam-] Trastuzumab Deruxtecan (DS-8201a), for HER2 Cancer Therapy. Chem Pharm Bull (Tokyo). 2019;67(3):173-185. doi: 10.1248/cpb.c18-00744. PMID: 30827997.
[4] Denevault-Sabourin, Caroline & Joubert, Nicolas & beck, alain & dumontet, charle. (2020). Antibody- Drug Conjugates: The Last Decade. Pharmaceuticals. 13. 245. 10.3390/ph13090245.
[5] Joubert N, Beck A, Dumontet C, Denevault-Sabourin C. Antibody-Drug Conjugates: The Last Decade. Pharmaceuticals (Basel). 2020 Sep 14;13(9):245. doi: 10.3390/ph13090245. PMID: 32937862; PMCID: PMC7558467.
[6] Pahl A, Lutz C, Hechler T. Amanitins and their development as a payload for antibody-drug conjugates. Drug Discov Today Technol. 2018 Dec;30:85-89. doi: 10.1016/j.ddtec.2018.08.005. Epub 2018 Sep 24. PMID: 30553524.
[7] Li JY, Perry SR, Muniz-Medina V, Wang X, Wetzel LK, Rebelatto MC, Hinrichs MJ, Bezabeh BZ, Fleming RL, Dimasi N, Feng H, Toader D, Yuan AQ, Xu L, Lin J, Gao C, Wu H, Dixit R, Osbourn JK, Coats SR. A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy. Cancer Cell. 2016 Jan 11;29(1):117-29. doi: 10.1016/j.ccell.2015.12.008. Erratum in: Cancer Cell. 2019 Jun 10;35(6):948-949. PMID: 26766593.
[8] Su D, Zhang D. Linker Design Impacts Antibody-Drug Conjugate Pharmacokinetics and Efficacy via Modulating the Stability and Payload Release Efficiency. Front Pharmacol. 2021 Jun 23;12:687926. doi: 10.3389/fphar.2021.687926. PMID: 34248637; PMCID: PMC8262647.
[9] Zhang D, Yu SF, Khojasteh SC, Ma Y, Pillow TH, Sadowsky JD, Su D, Kozak KR, Xu K, Polson AG, Dragovich PS, Hop CECA. Intratumoral Payload Concentration Correlates with the Activity of Antibody-Drug Conjugates. Mol Cancer Ther. 2018 Mar;17(3):677-685. doi: 10.1158/1535-7163.MCT-17-0697. Epub 2018 Jan 18. PMID: 29348271.
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