Beyond Expression: Functional Biology in ADC Target Evaluation

Introduction: ADC Target Evaluation Is Becoming Increasingly Biology-Driven

Over the past decade, antibody-drug conjugates (ADCs) have emerged as one of the fastest-growing therapeutic modalities in oncology. As of early 2026, more than 20 ADCs have received global regulatory approval across hematologic malignancies and solid tumors, expanding treatment options in diseases including breast cancer, gastric cancer, urothelial carcinoma, cervical cancer, and lung cancer.

Figure 1. Global list of approved ADC therapeutics as of early 2026.

Early ADC development largely prioritized tumor-associated antigens with high surface expression and limited normal tissue distribution to enable selective payload delivery. However, accumulating clinical and translational evidence suggests that expression level alone is often insufficient to predict ADC efficacy, safety profile, or durability of response.

ADC performance is now increasingly evaluated in the context of multiple biological parameters, including antigen accessibility, internalization efficiency, intracellular trafficking behavior, antigen shedding, tumor heterogeneity, and microenvironmental context. As competition around clinically validated targets such as HER2 and Trop-2 continues to intensify, ADC target evaluation is gradually expanding beyond expression profiling toward broader functional and translational assessment.

Rethinking Target Specificity: Balancing Expression and Therapeutic Window

Historically, the ideal ADC target was defined as an antigen with high and homogeneous tumor expression, minimal normal tissue expression, stable membrane localization, and efficient internalization capability. In practice, however, truly tumor-exclusive antigens are uncommon.

Figure 2. Overview of ADC mechanism of action. Adapted from Wang et al., Front. Immunol. 2025.

Clinically validated targets such as TROP-2, Nectin-4, and HER2 are also expressed in normal epithelial tissues, contributing to on-target/off-tumor toxicities including skin toxicity, ocular adverse events, and interstitial lung disease (ILD). Consequently, ADC development has increasingly focused on optimizing therapeutic window rather than pursuing absolute tumor specificity alone.

Trastuzumab deruxtecan (Enhertu®) provides a representative example of this evolving evaluation framework. Clinical activity observed in HER2-low and HER2-ultralow tumors suggests that very high antigen density may not always be required for meaningful ADC activity. In these settings, payload potency, linker design, drug-to-antibody ratio (DAR), and bystander killing capability may collectively contribute to activity despite lower target expression levels.

Figure 3. Comparison of selected characteristics between Kadcyla® and Enhertu®. Adapted from Esapa B. et al., Cancers (Basel). 2023.

At the same time, researchers are increasingly evaluating context-dependent antigen accessibility. Claudin18.2 (CLDN18.2) represents a notable example. In healthy gastric tissue, CLDN18.2 is largely confined within tight junctions and remains poorly accessible to circulating antibodies. During malignant transformation, loss of epithelial polarity exposes these epitopes, potentially enabling selective tumor targeting despite detectable expression in normal tissues.

These observations highlight that antigen accessibility and tissue context may substantially influence ADC targetability alongside expression level itself.

Internalization Dynamics Directly Influence Payload Delivery

Beyond target expression, productive intracellular delivery remains a central requirement for ADC activity. Following antigen engagement, the ADC-antigen complex typically undergoes internalization and intracellular trafficking to enable linker cleavage and payload release. Accordingly, internalization kinetics and trafficking behavior have become increasingly important considerations during ADC target evaluation.

Some hematologic malignancy targets, including CD22 and CD33, exhibit highly efficient receptor-mediated endocytosis and lysosomal trafficking, making them favorable for ADC development. In contrast, many solid tumor targets display more complex intracellular trafficking behavior.

Some clinically relevant solid tumor targets, including HER2 and TROP-2, may undergo receptor recycling following endocytosis rather than efficient lysosomal degradation. This trafficking behavior may reduce intracellular payload release efficiency and potentially limit ADC activity in certain settings.

As a result, ADC discovery teams increasingly rely on functional assays capable of distinguishing recycling versus degradative trafficking pathways. Technologies including pH-sensitive fluorescent probes, live-cell imaging, and quantitative flow cytometry-based assays are becoming important tools for characterizing target internalization behavior and intracellular trafficking dynamics.

Importantly, internalization properties must also be evaluated together with linker chemistry and payload mechanism. Cleavable linker systems and membrane-permeable payloads may partially compensate for inefficient intracellular trafficking through bystander killing effects, whereas non-cleavable linker systems often depend more heavily on efficient lysosomal processing for optimal activity.

Antigen Shedding: A Biological Liability and Distribution Variable

Antigen shedding remains an important consideration in ADC development. Shedding occurs when the extracellular domain (ECD) of a membrane protein is proteolytically released into circulation. High-shedding targets such as HER2, MUC16, and mesothelin may generate soluble antigen capable of binding circulating ADC molecules before tumor engagement.

Figure 4. Proposed impact of antigen shedding on binding-site barrier effects and intratumoral ADC distribution. Adapted from Esapa B. et al., Cancers (Basel). 2023.

Traditionally, this phenomenon has been viewed primarily as a liability because it may reduce tumor delivery efficiency, alter pharmacokinetics, and contribute to peripheral "sink effects."

However, several studies have proposed that antigen shedding may also influence intratumoral distribution. In highly antigen-dense tumors, ADC penetration can be limited by the "binding-site barrier" effect, where ADC molecules bind rapidly to tumor cells near the vasculature, potentially restricting diffusion into deeper tumor regions.

Moderate antigen shedding may partially alleviate this barrier by reducing excessive peripheral binding and improving intratumoral distribution. Consequently, antigen shedding is increasingly being evaluated not only as a potential liability, but also as a biological variable that may influence affinity optimization, tissue penetration behavior, and dosing strategy.

Clinical and Platform Advances Continue to Expand ADC Target Evaluation

Clinical experience and advances in ADC platform engineering continue to influence how target suitability is evaluated.

One major trend is the increasing competition around clinically validated targets such as HER2 and TROP-2. The rapid expansion of follow-on ADC programs has increased interest in differentiation through linker design, payload selection, biomarker-defined patient segmentation, and safety profile optimization.

At the same time, emerging solid tumor targets including Claudin18.2 (CLDN18.2), DLL3, and B7-H3 are attracting growing attention because of their relatively restricted expression patterns, prevalence in difficult-to-treat tumors, and potentially favorable internalization properties.

Clinical activity observed in HER2-low settings has also expanded interest in targets with lower or heterogeneous expression profiles. Advances in linker chemistry, higher DAR formats, and membrane-permeable payloads with bystander killing capability have broadened the range of expression profiles considered potentially actionable for ADC development.

In parallel, multi-antigen approaches are receiving increasing attention. Beyond dual-specific ADC architectures such as EGFR×HER3 and HER2×HER3, scientists are also exploring dual-payload ADCs designed to simultaneously deliver mechanistically distinct cytotoxic agents. These approaches are being investigated as potential strategies to address tumor heterogeneity and treatment resistance associated with single-target dependency.

Figure 5. Key design considerations for dual-payload ADC development. Adapted from Tao J. et al., Eur J Med Chem. 2025.

Finally, ADC development is becoming increasingly precision-oriented. Biomarker-driven patient selection, spatial profiling, and molecular stratification are playing growing roles in identifying patient populations that may derive the greatest benefit from specific ADC therapies.

Emerging Directions in ADC Target Evaluation

Immune-related targets such as B7-H3 (CD276), PD-L1, and CD47 are attracting increasing interest because of their dual associations with tumor immune regulation and tumor cell survival pathways. In these contexts, ADCs are also being investigated for their potential influence on tumor immune microenvironments in addition to direct cytotoxic activity.

At the same time, several previously challenging targets---including ROR1, CEACAM5, and EphA2-are being re-evaluated due to advances in linker technology, payload chemistry, and biomarker stratification.

EphA2 represents a notable example. Although earlier monoclonal antibody programs targeting EphA2 demonstrated limited clinical success, its rapid receptor turnover and strong internalization behavior have renewed interest in ADC-based therapeutic strategies.

Together, these developments illustrate how ADC target evaluation criteria continue to evolve alongside advances in ADC platform design and translational understanding.

Supporting Functional ADC Target Evaluation

As ADC target evaluation increasingly incorporates functional and translational biological parameters, robust analytical tools are becoming important for reducing uncertainty during early-stage discovery and lead optimization.

To support ADC research workflows, ACROBiosystems provides an integrated portfolio of ADC-focused research solutions spanning target validation, internalization analysis, Fc characterization, and bioanalytical development.

Key capabilities include:

- Recombinant proteins covering major ADC-relevant targets including HER2, TROP-2, CLDN18.2, B7-H3, EGFR, and c-MET.

- Internalization detection reagent for evaluating endocytosis kinetics and intracellular trafficking

- FcRn and Fcγ receptor proteins for Fc-related characterization and stability assessment

- Anti-payload antibodies and ADA supporting PK, ADA, and CMC workflows

These tools support more comprehensive functional characterization during ADC discovery and translational evaluation.

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Conclusion

ADC target evaluation is increasingly moving beyond expression level alone toward broader assessment of target biology and functional compatibility.

Internalization behavior, intracellular trafficking, antigen accessibility, shedding characteristics, tumor heterogeneity, and biomarker-driven patient stratification may all influence ADC activity and therapeutic window.

As next-generation ADC platforms continue to evolve, successful ADC target evaluation will increasingly depend not only on expression profile, but also on whether target biology supports efficient payload delivery, acceptable therapeutic window, and durable anti-tumor activity.

FAQ

Q1: Is high antigen expression still required for effective ADC target selection?

A: High antigen expression is no longer the sole determinant of ADC success, although it remains an important baseline factor.

In modern ADC development, expression level is increasingly evaluated together with functional biological parameters, including internalization efficiency, intracellular trafficking behavior, antigen accessibility, and overall therapeutic window.

Clinical evidence from HER2-low ADCs further demonstrates that meaningful anti-tumor activity can still be achieved in tumors with low or heterogeneous antigen expression, particularly when supported by optimized linker-payload design and bystander killing effects.

Q2: Why is target internalization efficiency important for ADC efficacy?

A: Target internalization efficiency is critical because ADC efficacy depends not only on antigen binding, but also on successful intracellular delivery of the payload.

After binding, the ADC--antigen complex must undergo endocytosis and trafficking to lysosomes for effective payload release. However, different targets exhibit distinct trafficking behaviors: some are efficiently routed to lysosomes, while others recycle back to the cell surface after internalization.

These differences directly impact intracellular drug release efficiency and overall therapeutic activity. Therefore, internalization assays, live-cell imaging, and trafficking studies are widely used to evaluate ADC target suitability during early discovery and validation.

Q3: How does antigen shedding influence ADC pharmacokinetics and tumor delivery?

A: Antigen shedding can significantly influence ADC pharmacokinetics, tumor delivery, and intratumoral distribution.

When the extracellular domain of a membrane antigen is cleaved and released into circulation, it may bind circulating ADC molecules, creating a "sink effect" that reduces the amount of drug available for tumor targeting.

However, antigen shedding may also have context-dependent effects on tumor penetration. In some cases, moderate shedding can reduce excessive perivascular binding and partially alleviate the binding-site barrier effect, potentially improving intratumoral distribution.

Q4: What experimental tools are used to evaluate ADC target biology and function?

A: Functional ADC target evaluation relies on a combination of biochemical, cellular, and bioanalytical tools designed to characterize both target biology and drug interaction properties.

These typically include recombinant target proteins for binding and screening studies, cell-based internalization assays to assess endocytic behavior, FcRn and Fcγ receptor proteins for Fc-related characterization, and anti-payload antibodies for pharmacokinetic (PK), anti-drug antibody (ADA), and bioanalytical assay development.

Together, these tools enable a more comprehensive understanding of target suitability beyond expression level alone.

Q5: Why are CLDN18.2, B7-H3, and DLL3 considered promising emerging ADC targets?

A: CLDN18.2, B7-H3, and DLL3 are considered promising emerging ADC targets due to their favorable biological and clinical characteristics.

These targets typically exhibit restricted expression in normal tissues, increasing their potential therapeutic window in ADC development. They are also highly relevant in difficult-to-treat tumor types with limited targeted therapy options.

In addition, their tumor biology may support ADC development: for example, CLDN18.2 becomes more accessible following loss of epithelial polarity in malignant tissues, while B7-H3 and DLL3 are associated with tumor contexts where novel targeted approaches are still needed.