Evolving ADC Payloads and Linkers: A Quick Guide

Antibody-drug conjugates (ADCs) are among the most actively engineered therapeutic formats in oncology, combining the targeting precision of monoclonal antibodies with the cytotoxic potency of small-molecule payloads. As the field moves beyond first-generation designs, the selection and optimization of payloads and linkers has become the central engineering challenge separating clinical success from dose-limiting failure.

For scientists, program leads, and business development professionals working on next-generation ADC programs, the decisions covered here, from payload mechanism selection to linker chemistry and tumor microenvironment (TME) activation strategies, are among the most consequential in ADC design.

What makes payload selection so critical in ADC engineering?

Payload potency must compensate for the fact that only approximately 2% of an infused ADC dose reaches the tumor site. This delivery inefficiency means that payloads must be exceptionally cytotoxic, stable under lysosomal conditions, and mechanistically matched to the tumor biology being targeted. Selecting the wrong payload class, regardless of how well the antibody performs, can result in insufficient tumor kill, off-target toxicity, or resistance emergence.

The three dominant payload mechanisms in clinical use are microtubule inhibition, DNA damage, and topoisomerase I (TOP1) inhibition. Each offers distinct advantages: microtubule inhibitors such as MMAE and DM4 are well-characterized and compatible with established linker chemistries, while TOP1 inhibitors such as deruxtecan (DXd) and exatecan are gaining ground due to their strong bystander effect, which extends cytotoxic reach into antigen-negative tumor cells within heterogeneous solid tumors.

Which payload classes dominate late-stage ADC clinical programs?

TOP1 inhibitors have become the leading payload class in Phase 3 ADC trials, representing approximately 46% of candidates at that stage. This is a significant increase compared to their 12% share among FDA-approved ADCs, reflecting a deliberate shift toward next-generation ADC payloads that perform well in solid tumor settings where antigen expression is variable.

TOP1 inhibitor payloads are increasingly represented in late-stage ADC development, with examples including adizutecan, DXd (deruxtecan), exatecan, KL610023, P1003, SHR9265, and SN-38. Microtubule inhibitors, including MMAE and MMAF, remain widely used but are no longer the sole dominant class. Primary payload categories in late-stage development are summarized here:

What are emerging payload classes and why are they attracting investment?

Three emerging payload categories are expanding what ADCs can achieve beyond direct cytotoxicity. Immune-activating payloads, including STING and TLR agonists delivered as immune-stimulating antibody conjugates (ISACs), convert immunologically cold tumors into inflamed environments by activating myeloid cells, enhancing T-cell recruitment, and promoting Fcγ receptor-mediated phagocytosis. Candidates including XMT2056, DS3610, and TAX500 have entered Phase 1 trials.

RNA-inhibitory payloads such as alpha-amanitin and triptolide offer a mechanistically distinct advantage: the ability to kill both actively dividing and dormant tumor cells, addressing a key resistance mechanism that cytoskeletal and topoisomerase-targeting agents cannot overcome. At least two antibody conjugates using RNA polymerase II inhibitor payloads, HDP101 and KH815, have entered clinical evaluation, with KH815 representing a first-in-class dual-payload design combining exatecan and triptolide.

BCL-XL inhibitors represent a third emerging class aimed at tumors that have upregulated anti-apoptotic pathways. Systemic BCL-XL inhibition has historically been limited by thrombocytopenia and cardiovascular toxicity, but antibody-mediated delivery offers a path to tumor-selective use. ABBV155 (mirzotamab clezutoclax) is the first ADC in this category to reach clinical trials.

What is the difference between cleavable and non-cleavable linkers, and when does each apply?

Cleavable vs. non-cleavable linkers are engineered to respond to specific intracellular or extracellular cues, including pH changes, redox conditions, or protease activity, releasing the payload only under defined biological conditions. Non-cleavable linkers, by contrast, require complete lysosomal degradation of the antibody before the active drug-linker catabolite is liberated. This distinction has direct consequences for bystander killing and off-target toxicity.

Non-cleavable linkers offer superior plasma stability but limit membrane permeability of the released catabolite, reducing bystander activity in heterogeneous tumors. This trade-off is reflected in clinical adoption: non-cleavable linkers account for approximately 18% of FDA-approved ADCs and only around 8% of late-stage candidates. Cleavable Val-Cit peptide and GGFG tetrapeptide linkers continue to predominate because they provide predictable, tumor-activated release while supporting the bystander killing needed in solid tumor settings.

What are the known limitations of Val-Cit linkers and how are developers addressing them?

Traditional Val-Cit-PAB linkers are the most widely used cleavable chemistry in approved ADCs, but they carry documented vulnerabilities. Susceptibility to premature cleavage by plasma enzymes, including Ces1C and neutrophil elastase, can trigger early payload release and dose-limiting toxicities. Aggregation at higher drug-to-antibody ratios (DARs) and hydrophobicity-driven pharmacokinetic variability are additional constraints.

Several engineering strategies are addressing these limitations. Incorporating hydrophilic scaffolds such as PEG spacers or modified peptide sequences (Glu-Val-Cit, Glu-Gly-Cit) reduces enzymatic sensitivity and improves aqueous stability. A more recent approach repositions the Val-Cit motif to the exo-position of the p-aminobenzyl carbamate (PAB) moiety, producing “exo-linkers” with enhanced hydrophilicity, reduced aggregation, and improved intracellular release profiles. These modifications preserve the core cleavage mechanism while addressing the pharmacokinetic liabilities that have constrained first-generation designs.

Why does site-specific conjugation matter for ADC performance?

Heterogeneous conjugation, produced by traditional lysine- or cysteine-based methods, generates ADC populations with variable DAR and inconsistent pharmacokinetics. This variability translates directly into unpredictable efficacy and safety profiles, complicating dose optimization and regulatory characterization. Site-specific conjugation strategies address this by producing homogeneous ADC populations with defined DAR values.

Microbial transglutaminase (MTG) enables precise conjugation at Q295 in the Fc region, though this typically requires modification of the nearby N297 glycan. A newer approach pairs MTG with a minimal RKAA peptide linker, enabling direct conjugation to native antibodies and producing homogeneous DAR2 ADCs without altering Fc glycosylation. Compared to conventional stochastic conjugation, site-specific methods improve batch-to-batch consistency, reduce the proportion of over-conjugated species that drive toxicity, and support more predictable clinical translation.

What are TME-cleavable linkers and why are they relevant to solid tumor ADCs?

TME-cleavable linkers are designed to release their payload extracellularly within the tumor microenvironment, bypassing the requirement for ADC internalization. These linkers exploit enzymes enriched in the TME, including matrix metalloproteinases, cathepsins, beta-glucuronidase, and serine proteases such as urokinase plasminogen activator (uPA), to trigger payload release directly at the tumor site.

This extracellular release mechanism addresses two of the most persistent barriers in solid tumor ADC development: antigen heterogeneity and poor tumor penetration. Because payload release does not depend on antigen-positive cells internalizing the ADC, TME-cleavable designs can achieve bystander killing across antigen-negative subpopulations and penetrate dense stromal compartments that limit conventional ADC access. Nona Biosciences has developed proprietary TME-cleavable linker technology that enables extracellular payload release driven by tumor-intrinsic enzymes, and this approach is being advanced in discovery and preclinical programs targeting challenging solid tumor settings.

What is a dual-payload ADC and what problem does it solve?

A dual-payload ADC carries two mechanistically distinct cytotoxic agents on a single antibody scaffold, delivering both to the same tumor target simultaneously. The rationale is resistance prevention: tumors can develop resistance to a single payload’s mechanism of action, allowing resistant subpopulations to survive and drive regrowth. Delivering two payloads with different mechanisms of action broadens tumor clearance and reduces the probability that any single resistance mechanism will be sufficient to rescue the tumor cell population.

KH815, currently in clinical evaluation, exemplifies this approach by combining exatecan (a TOP1 inhibitor) with triptolide (an RNA polymerase II inhibitor), representing a first-in-class dual-payload design. Beyond resistance prevention, dual-payload ADCs also offer potential advantages in heterogeneous tumors where different subpopulations may be differentially sensitive to distinct cytotoxic mechanisms. This format is one of several increasingly sophisticated ADC architectures, alongside bispecific ADCs, that reflect the field’s broader shift toward combination-in-one-molecule strategies.

How does Nona Biosciences support ADC discovery and development?

Nona Biosciences provides end-to-end ADC discovery and development capabilities built on its Harbour Mice® (transgenic mice engineered to produce fully human heavy-chain-only antibodies) platform and integrated engineering services. Harbour Mice® generate both fully human HCAbs and conventional H2L2 antibodies with exceptional specificity and optimal affinity for tumor-associated antigens, providing the antibody component that underpins effective ADC design. With over 300 antibody discovery programs completed and 19+ clinical-stage molecules, the platform has been validated across multiple therapeutic contexts.

Beyond antibody discovery, Nona offers ADC engineering and conjugation capabilities covering conjugation strategy selection, payload pairing, linker design, and analytical characterization. This integrated workflow addresses the full chain of ADC optimization, from binder identification through developability assessment and preclinical evaluation. Nona’s proprietary TME-cleavable linker technology adds a differentiated capability for developers targeting solid tumors where conventional internalization-dependent release is insufficient. Partnering with Nona provides access to a fully integrated discovery-to-IND pathway through the Idea toward IND (I-to-I®) (Nona’s integrated end-to-end service pathway from ideation through IND filing) framework, reducing handoff risk and accelerating program timelines.

For developers working on next-generation ADC formats, including bispecific ADCs and dual-payload constructs, Nona’s bispecific and multispecific engineering capabilities extend the platform into these more complex architectures. To discuss how Nona’s ADC discovery and linker-payload expertise can support a specific program, reach out to the Nona Biosciences team directly.

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