Next-Generation Dual-Payload ADCs: Target Selection, Payload Synergy, and Clinical Pipeline Analysis

Introduction: Emerging Interest in Dual-Payload ADCs in Oncology Development

The clinical success of newer-generation antibody–drug conjugates (ADCs), particularly HER2-targeted agents such as trastuzumab deruxtecan (T-DXd, DS-8201), has established topoisomerase I inhibitors (TOP1i) as an important payload class in solid tumor treatment.

Despite these advances, single-payload ADCs targeting HER2, TROP2, Nectin-4, and other validated antigens continue to face challenges related to acquired resistance and intratumoral heterogeneity. Mechanisms associated with resistance include drug efflux (e.g., MDR1/BCRP), antigen downregulation, lineage plasticity, and activation of DNA damage response pathways. In addition, heterogeneous antigen expression and variable internalization efficiency across tumor subclones may result in differences in intracellular payload exposure and sensitivity.

These limitations have driven growing interest in dual-payload ADCs (also referred to as dual-toxin ADCs). By incorporating two payloads with distinct mechanisms of action within a single ADC molecule, dual-payload designs aim to address multiple biological mechanisms that may contribute to resistance while potentially improving activity across heterogeneous tumor cell populations.

As of 2026, multiple dual-payload ADC programs have advanced into IND-enabling, clinical, or early-stage translational development. Interest in this field was further reflected at AACR 2026, where an increasing number of dual-payload ADC programs and enabling conjugation technologies were presented.

Figure 1. Proposed mechanisms by which dual-payload ADCs may address tumor heterogeneity and resistance-associated biological processes. Source: Tao et al., Eur J Med Chem, 2025.

Payload Design Approaches in Dual-Payload ADCs

TOP1 inhibitors (TOP1i) continue to dominate the payload landscape and remain the backbone of most dual-payload ADC programs currently advancing through preclinical and clinical development. Current dual-payload strategies are largely differentiated by the selection of the second payload, with DNA damage response (DDR) inhibitors, immune-stimulatory agents, and transcriptional regulators representing some of the most actively explored categories.

Figure 2. Representative dual-payload ADC designs. (A) Cytotoxic and immunostimulatory payload combination. (B) Dual-cytotoxic payload combination. Source: Ravenstijn et al., Clin Transl Sci, 2026.

One of the most widely investigated approaches combines TOP1 inhibitors with DDR modulators, particularly ATR inhibitors. The rationale is to pair TOP1i-induced DNA damage with suppression of DNA repair pathways, thereby enhancing the biological consequences of DNA damage and potentially overcoming mechanisms associated with reduced sensitivity to TOP1 inhibition.

Another emerging strategy involves combining cytotoxic payloads with immune-stimulatory agents. Examples include TLR7/8 agonists and other immune-modulating payloads that are being evaluated alongside conventional cytotoxic mechanisms. These approaches seek to expand ADC activity beyond direct tumor cell killing, although clinical experience remains limited.

In parallel, increasing attention is being directed toward payloads that may influence components of the tumor microenvironment. Such approaches reflect broader efforts to address the biological complexity of heterogeneous tumors through payload diversification.

Beyond dual-payload designs, interest is also beginning to extend toward higher-order payload architectures. Early studies have reported triple-payload ADC concepts. One example is the AraLinQ™ platform developed by Araris Biotech, which has been used to generate a triple-payload ADC incorporating two TOP1 inhibitors together with MMAE. Although these approaches remain at an early stage, they illustrate continuing efforts to expand the range of payload combinations that can be incorporated within a single ADC.

Collectively, these developments highlight the increasing diversification of ADC payload design and the growing emphasis on combining distinct biological functions within a single therapeutic modality.

Linker Design and Payload Release

As payload combinations become more complex, linker design is becoming an increasingly important determinant of how individual payloads are released and function within dual-payload ADC systems.

In dual-payload ADCs, linker design plays an important role in controlling the release of individual payloads. Because the two payloads may differ in their mechanisms of action and intended sites of activity, linker selection can influence how and where each payload is released.

KH815, a TROP2-targeted dual-payload ADC, illustrates this approach. In this construct, Exatecan is conjugated through a lysosomal enzyme-cleavable linker, enabling intracellular release following ADC internalization. The second payload, a triptolide-derived RNA polymerase II inhibitor, is linked through a pH-sensitive linker designed to release payload under acidic conditions.

As a result, the two payloads exhibit different release profiles. While one payload is primarily released following intracellular processing, the second may be released under acidic extracellular conditions. The contribution of these release characteristics to overall antitumor activity remains under investigation.

Figure 3. Mechanism of action of KH815 Source: Chengdu Kanghong, publicly available program materials.

For dual-payload ADCs, linker design is therefore important not only for payload stability and release, but also for controlling payload ratio, DAR distribution, and overall molecular properties.

Potential applications of differentiated linker strategies include:
- Combining payloads with distinct mechanisms of action
- Enabling different release profiles for individual payloads
- Potentially improving payload exposure across heterogeneous tumor cell populations
- Supporting optimization of payload ratio and DAR distribution

The Emerging Dual-Payload ADC Target Landscape

As dual-payload ADCs continue to advance toward clinical development, several targets have emerged as major areas of investigation. Current programs are primarily concentrated on clinically validated ADC targets, including TROP2 and HER2, while development is also expanding into targets such as PTK7 and CEACAM5. Across these targets, payload selection is increasingly being tailored to challenges such as resistance to existing ADCs and tumor heterogeneity.

TROP2: A Leading Target in Dual-Payload ADC Development

TROP2 has become one of the most active targets in dual-payload ADC research. The clinical success of sacituzumab govitecan and datopotamab deruxtecan established TROP2 as a validated ADC target, while resistance observed following TOP1 inhibitor-based treatment has provided a rationale for incorporating additional payload mechanisms.

Several TROP2-targeted dual-payload ADCs have entered clinical or preclinical evaluation. KH815 combines exatecan with a triptolide-derived RNA polymerase II inhibitor, whereas CBB-120 combines a TOP1 inhibitor with an ATR inhibitor. Although these programs utilize different payload combinations, both incorporate mechanisms beyond TOP1 inhibition alone.

Current TROP2 programs remain largely centered on TOP1 inhibitor-containing payload combinations, with increasing interest in DNA damage response modulation and transcription-related targets.

HER2: Expanding Dual-Payload Development Beyond T-DXd

HER2 remains one of the most extensively validated ADC targets in oncology. Following the clinical success of trastuzumab deruxtecan, multiple groups have begun evaluating dual-payload approaches in HER2-directed ADCs, particularly in settings where mechanisms beyond TOP1 inhibition are being explored.

Examples include CLIO-8221, which combines exatecan with an ATR inhibitor, and IMD526, which incorporates a TOP1 inhibitor together with a TLR7/8 agonist. In parallel, several HER2-targeted constructs are evaluating combinations of TOP1 inhibitors and microtubule inhibitors using site-specific conjugation technologies.

PTK7 and CEACAM5: Emerging Targets for Dual-Payload ADC Development

While most dual-payload programs remain concentrated on clinically validated ADC targets such as TROP2 and HER2, development activity is increasingly extending to additional targets associated with heterogeneous solid tumors and broader patient populations.

PTK7 has attracted considerable interest because of its expression across multiple tumor types, including NSCLC, TNBC, ovarian cancer, and gastric cancer. This broad expression profile has made PTK7 an attractive candidate for evaluating whether dual-payload strategies can improve activity across biologically diverse tumor populations. STRO-227 represents one example of this approach, combining exatecan and MMAE within a PTK7-targeted ADC.

A similar trend is emerging in gastrointestinal malignancies. CEACAM5 has become an important ADC target in colorectal cancer and other gastrointestinal tumors, creating opportunities to evaluate dual-payload strategies beyond the more established HER2 and TROP2 settings. IBI3020 is among the earliest CEACAM5-targeted dual-payload ADCs to enter clinical evaluation and combines a topoisomerase inhibitor with MMAE.

Together, these programs illustrate how dual-payload development is gradually expanding from a small number of validated ADC targets toward a broader range of tumor-associated antigens.

Bispecific Target Combinations in Dual-Payload ADC Development

Target diversification is also emerging as an important area of innovation alongside payload diversification. In addition to single-target ADCs, several groups are exploring dual-payload strategies within bispecific antibody formats.

These constructs combine recognition of two tumor-associated antigens with delivery of two payload mechanisms in a single molecule. Examples include BCG048, which targets both ITGB6 and B7-H3, and IBI3028, which combines a bispecific antibody framework with TOP1 inhibitor and MMAE payloads using the DuetTx platform.

Although clinical experience remains limited, these programs demonstrate how dual-payload development is increasingly being integrated with multi-target ADC design strategies.

Clinical-Stage Dual-Payload ADC Landscape

Bioanalytical Complexity in Dual-Payload ADC Development

Dual-payload ADCs introduce increased analytical demands compared with conventional single-payload formats.

Key analytical requirements include quantification of multiple payload species, differentiation of conjugated and free drug fractions, assessment of DAR distribution, and evaluation of anti-drug antibody interference.

Key challenges include:
- instability and premature payload release
- DAR heterogeneity affecting pharmacokinetics
- manufacturing and scale-up constraints
- increased complexity in PK and ADA assay design
- potential cross-reactivity arising from structurally related payloads

These challenges are particularly relevant in bioanalytical development, where structurally similar payloads can complicate assay specificity and quantification accuracy.

To support these requirements, highly specific anti-payload antibody panels covering DXd, Exatecan, SN-38, MMAE, MMAF, and triptolide-derived payloads are increasingly applied.

These reagents are used in:
- PK characterization of dual-payload ADCs
- free payload quantification
- ADA assay development
- multi-payload bioanalysis workflows

As dual-payload ADCs become more complex, bioanalytical strategy is increasingly being considered alongside payload, linker, and conjugation design during development.

Conclusion

Dual-payload ADCs are emerging as an active area of ADC innovation, with increasing numbers of programs advancing into clinical development. Current efforts extend beyond simply combining two cytotoxic agents and increasingly focus on how target selection, payload pairing, and conjugation strategies influence therapeutic performance.

As clinical data continue to accumulate, the field will gain a clearer understanding of where dual-payload approaches can provide meaningful advantages over conventional single-payload ADCs across different tumor settings.

FAQ

Q1: Why are dual-payload ADCs attracting increasing attention in oncology drug development?

A: Dual-payload ADCs are being investigated as a potential strategy to address two major challenges in ADC development: acquired resistance and tumor heterogeneity. By incorporating two payloads with distinct mechanisms of action into a single ADC, these designs aim to expand biological activity beyond what may be achievable with a single payload alone. Current approaches include combinations involving TOP1 inhibitors, DDR inhibitors, immune agonists, and microtubule inhibitors. As more programs advance into clinical development, dual-payload ADCs are becoming an increasingly active area of research for next-generation targeted therapies.

Q2: What factors should be considered when selecting a target for a dual-payload ADC?

A: Target selection involves more than antigen expression alone. Internalization efficiency, expression heterogeneity, tumor distribution, and biological relevance can all influence ADC performance. These factors are particularly important when evaluating whether a dual-payload strategy may provide advantages over conventional ADC designs. To support target validation and antibody screening, ACROBiosystems offers recombinant proteins covering a broad range of ADC-relevant targets, including HER2, TROP2, CEACAM5, PTK7, B7-H3, Nectin-4, and EGFR family targets, enabling binding characterization and early-stage discovery studies.

Q3: How are payload combinations typically evaluated during dual-payload ADC development?

A: Selecting payloads with complementary biological activities is a key challenge in dual-payload ADC design. Researchers often assess target binding, internalization behavior, and downstream cellular responses before advancing candidate molecules. These studies help determine whether a payload combination may provide advantages in specific biological contexts. ACROBiosystems supports these workflows with recombinant target proteins, fluorescently labeled proteins, and ADC Internalization Detection Reagents that can be used to evaluate target engagement and intracellular uptake during ADC research and development.

Q4: Why are anti-payload antibodies becoming increasingly important for dual-payload ADC bioanalysis?

A: Dual-payload ADCs introduce additional complexity in pharmacokinetic (PK), anti-drug antibody (ADA), and free-payload analyses because multiple payload species must be monitored simultaneously. Assay specificity can become particularly challenging when structurally related payloads are involved. To support these studies, ACROBiosystems offers anti-payload antibodies covering commonly used ADC payload classes, including DXd, Exatecan, SN-38, MMAE, MMAF, and triptolide-derived payloads. These reagents can support PK assay development, free payload quantification, ADA assessment, and other bioanalytical workflows for next-generation ADC programs.