Antibody-drug conjugates payloads: then, now and next

Antibody-drug conjugates (ADCs) have been a groundbreaking approach to cancer treatment with their ability to deliver cytotoxic drugs directly to diseased cells while sparing healthy tissues. This targeted delivery allows enhanced therapeutic efficacy while reducing the harmful side effects commonly associated with traditional chemotherapy.

Here, we reflect on how ADCs and their payloads have changed, take a look at some of the less common payloads being explored in the treatment of cancer, and investigate the potential of dual-drug ADCs.

The evolution of ADC payloads

The evolution of ADCs and their payloads is a story of continuous innovation to counter the complexities of cancer. In the first generation, ADCs were paired with traditional chemotherapeutic agents like methotrexate, vinblastine and doxorubicin as cytotoxic payloads.¹

The research community pressed on, turning to novel compounds that were substantially more potent than those used in the first generation. Second-generation ADCs, with payloads like the tubulin polymerisation inhibitors maytansinoids and auristatins, stood out with their antiproliferative activity against tumour cells but also came with severe side effects. While highly effective against proliferating tumour cells, ADCs with antimitotic payloads typically struggled against slower-growing cancer types.²

In response, DNA-damaging agents that could target the entire cell cycle received renewed attention as ADC payloads. These agents, including enediynes, topoisomerase I inhibitors, duocarmycins and pyrrolobenzodiazepines (PBD) dimers, proved effective in providing a wider range of therapeutic options to tackle cancer.³ Despite this progression, ADCs still need to strike a balance between having potent cytotoxic payloads and minimising the side effects while hopefully overcoming concerns around drug resistance.⁴

But the horizon of ADCs is expanding even further, with the design of complex payloads. Groundbreaking strategies like proteolysis-targeting chimeric molecules (PROTACs) are also being explored.⁶ Combining the effect of payloads with different mechanisms of action – an approach that revolutionised small molecule chemotherapy – also holds the promise of enhanced therapeutic activity for ADCs. Dual cytotoxic payload ADCs could address the inherent heterogeneity in tumours. Associating immunomodulatory and cytotoxic payloads on the same ADC could also amplify the therapeutic effect of cytotoxic payloads harnessing the power of the immune system to target tumour cells.⁵ These novel approaches are charting the course for the next steps in the evolution of ADCs towards increased therapeutic efficacy and long-term patient survival rates.

Novel mechanisms of action 

As the processes within tumours become better understood, new areas to target are discovered. Some researchers are now investigating ways to interrupt protein synthesis and catabolism within tumours. More than ever, however, the payloads require careful design to avoid affecting surrounding tissues.

HSP90 inhibitors

HSP90, a major chaperone protein often overexpressed in tumours, has been targeted by inhibitors derived from the geldanamycin backbone.⁷ These inhibitors have faced challenges such as dose-limiting toxicity and poor pharmacokinetics, but geldanamycin ADCs have demonstrated increased survival in mice. Recent efforts are breathing new life into geldanamycin through the construction of a fusion protein made with an anti-HER2 scFv and an HSP90 inhibitor-binding domain (HER2 scFv-HBD), which has resulted in improved anti-cancer efficacy in HER2-positive cancers when loaded with geldanamycin derivatives.⁸

Translation inhibitors

Developing translation inhibitors will always be challenging due to their critical effects on healthy tissues, but omacetaxine was approved by the US Food and Drug Administration (FDA) in 2012 for the treatment of adult patients with chronic myeloid leukaemia.⁹ Others, like psymberin, have been used as ADC payloads, with a β-glucuronide linker to target CD-30-positive and CD-70-positive malignancies.¹⁰

Proteasome inhibitors

Since the approval of Bortezomib in 2003,¹¹ new proteasome inhibitors have emerged with reduced side effects. Carmaphycin B analogues, successfully conjugated to trastuzumab, although promising, have proven less potent than other ADCs, emphasising the tricky balance between potency and safety.¹²

PROTACs

PROTACs form part of an exciting addition to the ADC family: the degrader-antibody conjugates (DACs). Rather than inhibition, PROTACs trigger degradation by the proteasome with advantages including prolonged effects and potent cytotoxicity.¹³

Immune modulators as payloads

When we talk about ADC payloads, we usually refer to cytotoxic agents and their direct cancer cell-killing effects. Another effect that certain classes of payloads can elicit is immunogenic cell death (ICD).¹⁴ With these agents, the cell death process leads to the secretion of damage-associated molecular patterns (DAMPs), which activate the immune system, ultimately leading to further tumour destruction. More recently, researchers have begun to look at conjugating immunomodulatory agents to antibodies to directly activate an immune response against the tumour. 

This approach – immune-stimulating antibody conjugates (ISACs) – uses a payload that stimulates the innate and adaptive immune responses, recruiting tumour-fighting T cells.¹⁵ ISACs use small immuno-agonist molecules conjugated to the antibody to boost the immune response while minimising toxicity. Where traditional ADCs kill cancer cells directly, immune-modulating ADCs activate immune cells, allowing for more sophisticated targeting strategies and the potential to generate protective immune memory responses that may reduce future tumour cell re-growth or metastasis.

This oncology field is rapidly developing, with a variety of new immune-modulating ADC drugs under study. Current developments include payloads like Toll-like receptor (TLR) 7 or 8 agonists and stimulators of interferon gene (STING) molecules.^16–18 Overall, the combination of precise targeting and powerful modulation promises a new frontier in the battle against cancer.

Dual payload ADCs

As effective as therapies have been in treating solid and haematological cancers, tumour heterogeneity and resistance remain major clinical challenges. Tumour heterogeneity can lead to recurrence and metastasis, while acquired resistance can result in aggressive tumour growth and poor survival. To overcome these obstacles, combination therapy, which delivers multiple small molecules, has emerged as a potential solution.

One strategy being explored involves conjugating both drugs to the same linker. In a pioneering study, Levengood et al.¹⁹ developed one of the first dual-drug ADCs containing the antimitotic agents monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) connected to a single linker backbone. With this design, the resulting ADC combined the bystander activity of MMAE, which tackles issues related to heterogeneous target expression, with the higher potency of MMAF towards multi-drug resistant (MDR) cancer cells, owing to the lower susceptibility of this auristatin payload to efflux pumps. Notably, in resistant tumour models, while this dual-drug ADC showed potency comparable to single-drug ADCs, it achieved higher cure rates in vivo.

More recently, Yamazaki et al.²⁰ explored a dual-drug ADC using an alternative, flexible heterotrifunctional linker that facilitated precise control over drug-to-antibody ratios (DARs). Their MMAE/F dual-drug ADC with a DAR of 4+2 showed significant antitumour activity in mouse models of refractory breast cancer with heterogeneous HER2 expression. The team’s in vivo data indicated that dual-drug ADCs performed better than the co-administration of single-drug ADCs. 

The development of flexible dual-payload ADC synthesis platforms, like Yamazaki et al’s click chemistry modular assembly platform, will help with the generation of larger ADC libraries and speed up the screening process to identify effective combinations with diverse mechanisms of action. Ultimately, flexibility in dual-payload ADC development means better treatments could reach patients sooner.

Conclusion

ADCs have developed in leaps and bounds, shifting from simple payload structures to complex arrangements involving dual payloads, immune modulators, and innovative concepts like PROTACs. Pioneering research has led to the ability to simultaneously target multiple aspects of a tumour to deliver more effective treatment and overcome resistance. And of course, the introduction of immunomodulatory agents as payloads extends the potential of ADCs even further as it bridges direct cytotoxicity with immune system engagement for a more robust therapeutic response. These developments give researchers the tools to craft better therapeutics.

But challenges remain: at the core, ADCs must still balance potency with side effects. However, novel approaches like flexible synthesis platforms, combination therapies, and new classes of payloads such as HSP90, translation and proteasome inhibitors are the focus of ongoing research and could well be the next step in ADC evolution.

The future of ADCs looks bright, with potential for further innovation and refinement in design and application. If we are set on translating these scientific advances into tangible benefits for patients, then we must continue to push collaborations between research and clinical practice, as well make full use of our advanced analytical skills. Through the design of dual payloads, incorporation of immunomodulatory agents, and exploration of new molecular targets, the field of ADCs looks set to remain at the forefront of this new age of oncology and cancer therapy.

References

1. Trail PA, et al. Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates. Science 261, 212–215 (1993).

2. Florian S, Mitchison TJ. Anti-Microtubule Drugs. in The Mitotic Spindle: Methods and Protocols (eds. Chang, P. & Ohi, R.) 403–421 (Springer, 2016). doi:10.1007/978-1-4939-3542-0_25.

3. Fu Y, Ho M. DNA damaging agent-based antibody-drug conjugates for cancer therapy. Antib. Ther. 1, 33–43 (2018).

4. Collins DM, Bossenmaier B, Kollmorgen G, Niederfellner G. Acquired Resistance to Antibody-Drug Conjugates. Cancers 11, 394 (2019).

5. Gerber H-P, Sapra P, Loganzo F, May C. Combining antibody–drug conjugates and immune-mediated cancer therapy: What to expect? Biochem. Pharmacol. 102, 1–6 (2016).

6. Antibody–PROTAC Conjugates Enable HER2-Dependent Targeted Protein Degradation of BRD4 | ACS Chemical Biology. https://pubs.acs.org/doi/full/10.1021/acschembio.0c00285.

7. Park H-K, et al. Unleashing the full potential of Hsp90 inhibitors as cancer therapeutics through simultaneous inactivation of Hsp90, Grp94, and TRAP1. Exp. Mol. Med. 52, 79–91 (2020).

8. Lim KS, Lee DY, Han S, et al. Targeted delivery of heat shock protein 90 inhibitors prevents growth of HER2-positive tumor. Biomaterials 273, 120817 (2021).

9. Omacetaxine: A Protein Translation Inhibitor for Treatment of Chronic Myelogenous Leukemia | Clinical Cancer Research | American Association for Cancer Research. https://aacrjournals.org/clincancerres/article/20/7/1735/252368/Omacetaxine-A-Protein-Translation-Inhibitor-for.

10. Expanded Utility of the β-Glucuronide Linker: ADCs That Deliver Phenolic Cytotoxic Agents | ACS Medicinal Chemistry Letters. https://pubs.acs.org/doi/10.1021/ml100039h.

11. Kane RC, Farrell AT, Sridhara R, Pazdur R. United States Food and Drug Administration approval summary: bortezomib for the treatment of progressive multiple myeloma after one prior therapy. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 12, 2955–2960 (2006).

12. Almaliti J, et al. Exploration of the carmaphycins as payloads in antibody drug conjugate anticancer agents. Eur. J. Med. Chem. 161, 416–432 (2019).

13. Dragovich PS. Degrader-antibody conjugates. Chem. Soc. Rev. 51, 3886–3897 (2022).

14. Zhou J, et al. Immunogenic cell death in cancer therapy: Present and emerging inducers. J. Cell. Mol. Med. 23, 4854–4865 (2019).

15. Ackerman SE, et al. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nat. Cancer 2, 18–33 (2021).

16. Wu Y, et al. Tumor-targeted delivery of a STING agonist improves cancer immunotherapy. Proc. Natl. Acad. Sci. 119, e2214278119 (2022).

17. Conilh L, Sadilkova L, Viricel W, Dumontet C. Payload diversification: a key step in the development of antibody–drug conjugates. J. Hematol. Oncol.J Hematol Oncol 16, 3 (2023).

18. Duvall J R, et al. Discovery and Optimization of a STING Agonist Platform for Application in Antibody Drug Conjugates. J. Med. Chem. 66, 10715–10733 (2023).

19. Levengood MR, et al. Orthogonal Cysteine Protection Enables Homogeneous Multi-Drug Antibody–Drug Conjugates. Angew. Chem. Int. Ed. 56, 733–737 (2017).

20. Yamazaki C M, et al. Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance. Nat. Commun. 12, 3528 (2021).