Introduction

Antibody-drug conjugates (ADCs) have emerged as a transformative modality in oncology, enabling the targeted delivery of cytotoxic agents to tumor cells while minimizing systemic toxicity. However, their clinical success has been hampered by challenges such as the development of resistance, tumor heterogeneity, and suboptimal drug release. The research presented in Bioconjugate Chemistry (2025, 36, 190−202) DOI: 10.1021/acs.bioconjchem.4c00398 introduces a modular synthetic strategy to generate dual-drug ADCs that aim to address these limitations.

Background

Current ADCs are typically designed with a single cytotoxic payload, limiting their therapeutic window and increasing susceptibility to resistance mechanisms. HER2-positive tumors, which constitute a clinically aggressive subset of breast and gastric cancers, have been successfully targeted using trastuzumab-based ADCs such as T-DM1 (Kadcyla) and T-DXd (Enhertu) [1,2]. However, their efficacy is often compromised by tumor evolution, leading to resistance through antigen downregulation, altered intracellular trafficking, and efflux mechanisms [3].

To counteract these challenges, researchers from the University of Utah and AstraZeneca have developed a library of dual-drug ADCs, allowing simultaneous delivery of two mechanistically distinct cytotoxic agents. This modular synthesis strategy enables precise control over drug-to-antibody ratios (DAR), facilitating optimization of efficacy and toxicity profiles.

Methodology

The dual-drug ADCs were constructed on an anti-HER2 trastuzumab scaffold, incorporating two distinct conjugation strategies:

• Cyclopentadiene-containing unnatural amino acids were introduced into the antibody, allowing site-specific conjugation via a Diels-Alder reaction with maleimide-functionalized linker-drug constructs [4].

• Native cysteine residues were reduced and modified via thiol-maleimide chemistry, enabling a second site-specific conjugation [5].

This dual conjugation approach allowed researchers to generate 19 unique ADC constructs, systematically varying drug payload combinations, linker chemistries, and DARs.

Key Findings

1. Enhanced Cytotoxicity of Dual-Drug ADCs

The researchers evaluated their ADC library against HER2-positive and HER2-low cancer cell lines, revealing that several dual-drug ADCs exhibited superior cytotoxicity compared to their single-drug counterparts. Notably:

• T-DA2(MMAE)-Cys(VBL) demonstrated significantly improved potency, indicating a synergistic effect between the two microtubule-disrupting agents [6].

• T-DA2(VBL)-Cys(DMAG) exhibited enhanced activity, suggesting that HSP90 inhibition via geldanamycin analogs can potentiate ADC trafficking and drug release [7].

• T-DA4(DMAG)-Cys(Aldox) displayed increased toxicity in HER2-low expressing MDA-MB-453 cells, highlighting the potential for targeting tumors with heterogeneous HER2 expression [8].

2. Optimized Drug-to-Antibody Ratios

One of the major challenges in ADC design is balancing payload delivery without compromising pharmacokinetics or increasing off-target toxicity. The modular approach allowed researchers to fine-tune DARs, with constructs exhibiting total drug loadings ranging from 3.0 to 9.7. Importantly, higher DAR constructs maintained stability, demonstrating minimal aggregation over extended storage periods [9].

3. Overcoming ADC Resistance Mechanisms

Resistance to ADCs often arises from:

• Antigen downregulation, reducing ADC uptake [10].

• Efflux transporter upregulation, diminishing intracellular drug accumulation [11].

• Altered intracellular trafficking, preventing payload release [12].

By incorporating dual-payload strategies, the researchers observed improved retention and intracellular processing of their ADC constructs. Specifically, geldanamycin-conjugated ADCs facilitated HER2 degradation, reducing receptor recycling and increasing lysosomal trafficking—a crucial factor for efficient drug release [13].

Implications for Next-Generation ADCs

The findings of this study offer profound implications for ADC development:

• Combination Therapeutics within a Single ADC: The ability to co-deliver two mechanistically distinct payloads within a single ADC could reduce the need for combination regimens, simplifying treatment protocols [14].

• Targeting HER2-Heterogeneous Tumors: Enhanced efficacy in HER2-low expressing cells suggests that dual-drug ADCs could be valuable in expanding the patient population eligible for HER2-targeted therapies [15].

• Mitigating Drug Resistance: This modular approach provides a promising strategy to counteract resistance mechanisms that have historically limited the success of single-drug ADCs [16].

Future Directions

While in vitro studies highlight the advantages of dual-drug ADCs, further in vivo validation is necessary to assess pharmacokinetics, biodistribution, and therapeutic index. Future work will focus on:

• Evaluating tumor penetration and drug release kinetics in murine xenograft models [17].

• Investigating immune-modulatory effects of dual-payload ADCs [18].

• Exploring the applicability of this strategy to other tumor-associated antigens, beyond HER2 [19].

Conclusion

The development of modularly synthesized dual-drug ADCs represents a significant advancement in the field of targeted cancer therapy. By leveraging site-specific conjugation techniques, researchers have created a platform that enables systematic evaluation of drug combinations, optimizing both efficacy and safety. As ADC technology continues to evolve, dual-payload strategies may pave the way for more durable and broadly effective treatments for HER2-positive and HER2-low cancers.

 

 

References

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19. Black et al., Trends Pharmacol Sci. 2022.

For full experimental details, the complete study is available in Bioconjugate Chemistry (2025, 36, 190−202) DOI: 10.1021/acs.bioconjchem.4c00398.