The emergence of targeted drug delivery systems, particularly antibody-drug conjugates (ADCs) and peptide-drug conjugates (PDCs), marks the advent of a precision era in cancer therapy, akin to "biological missiles." The core design principle of these systems involves covalently linking a highly targeted carrier (such as an antibody or peptide) to a potent payload with strong cytotoxic activity via a chemical bridge called a "linker." The ultimate goal is to achieve specific drug accumulation and release at the lesion site, thereby maximizing therapeutic efficacy while minimizing systemic toxicity to healthy tissues. Within this intricate "trinity" structure, the selection and optimization of the linker and payload directly determine the stability, safety, pharmacokinetic properties, and ultimate therapeutic efficacy of the conjugated drug. Therefore, deep exploration of the chemical properties of linkers and the mechanisms of action of payloads, along with achieving a perfect balance between the two, has become a key driver propelling this field forward. This paper will systematically elaborate on the types of linkers and payloads, selection strategies, optimization challenges, and future development directions.
A successful targeted drug delivery system is far more than the simple sum of its parts. A dynamic and intricate interplay exists between the carrier, linker, and payload. The carrier is responsible for precise navigation, yet its efficacy ultimately depends on the payload's killing power. The linker, serving as the bridge between carrier and payload, directly determines the system's success or failure based on its performance. An ideal design demands that the linker maintain absolute stability during systemic circulation to prevent premature payload release, which could cause off-target toxicity and reduce drug delivery to the target site. However, once the conjugate enters the target cell via internalization, the linker must be cleaved efficiently and rapidly to release sufficient active payload for its pharmacological effect. Simultaneously, the payload itself must possess extremely high potency (typically at the nanomolar or even picomolar level), as each carrier can transport only a limited number of molecules. Furthermore, physicochemical properties of the payload, such as hydrophobicity, significantly influence the aggregate state, pharmacokinetics, and distribution characteristics of the entire conjugate. Therefore, the selection and optimization of linkers and payloads constitute an interconnected, mutually constrained systemic endeavor, representing a core element in enhancing the efficacy and safety of targeted therapeutics.
Linkers serve as "smart switches" that control the spatiotemporal precision of payload release. Based on their chemical structure and cleavage mechanisms in biological environments, linkers can be primarily categorized into two types: cleavable linkers and non-cleavable linkers. Each type possesses distinct advantages and applicable scenarios, with selection determined by the characteristics of the target antigen, internalization efficiency, and the desired scope of cytotoxic effects.
Cleavable linkers rely on biological differences between intracellular and extracellular environments to achieve specific cleavage. These linkers provide a trigger-release mechanism for conjugated drugs, serving as a key enabler for efficient intracellular drug delivery. Enzyme-catalyzed cleavable linkers represent one of the most widely used and successful types. Their design capitalizes on the presence of specific hydrolases (e.g., cathepsins, phospholipases) within tumor lysosomes. For instance, the valine-citrulline dipeptide linker serves as a classic cathepsin B-cleavable linker—stable in plasma but efficiently cleaved upon lysosomal internalization, releasing the active drug. Second, pH-sensitive linkers exploit the acidic tumor microenvironment and the further reduced pH (pH 4.5–5.0) within endosomes/lysosomes. The hydrazone bond is a representative pH-sensitive linker, stable in the neutral pH of the bloodstream but rapidly hydrolyzed in acidic lysosomes. Additionally, redox-sensitive linkers exploit the disparity in glutathione concentration between intracellular compartments (particularly the cytoplasm and mitochondria) and the extracellular environment. Linkers containing disulfide bonds cleave under the reducing action of high-concentration glutathione, enabling specific drug release within the cytoplasm. These linkers offer expanded possibilities for achieving precise drug delivery to specific cellular compartments.
In contrast to cleavable linkers, non-cleavable linkers lack the property of being cleaved by specific biological mechanisms in vivo. They are typically formed by highly stable chemical bonds, such as thioether bonds. Drugs conjugated with such linkers require complete degradation by lysosomes after entering target cells, ultimately releasing the payload metabolite still linked to the carrier amino acid residue. The primary advantage of this design lies in its exceptional plasma stability, which minimizes premature drug release before reaching the target site, thereby significantly reducing off-target toxicity. However, its limitation lies in the requirement that the released drug metabolite maintain activity comparable to the original payload. Additionally, its transmembrane capacity may be impaired, thereby restricting the "bystander effect"—the ability of the drug to be released from apoptotic target cells and kill neighboring tumor cells. Consequently, non-cleavable linkers are more suitable for cases where the target antigen is highly expressed, internalization efficiency is high, and tumor cells are uniformly distributed.
The payload serves as the "warhead" that delivers the ultimate killing effect in targeted delivery systems. Its selection criteria are extremely stringent, demanding not only exceptional efficacy but also compatibility with the linker chemistry. With technological advancements, PDC payload options have expanded beyond traditional cytotoxic drugs to encompass a broader range of therapeutic modalities.
Currently, most approved or investigational PDCs still employ small-molecule cytotoxic drugs as payloads. These agents represent potent chemotherapeutic drugs validated over decades of clinical use, characterized by well-defined mechanisms of action and strong cytotoxic potency. Key categories include: - Microtubule inhibitors, such as auristatin derivatives (MMAE, MMAF) and vinblastine derivatives (DM1, DM4), which inhibit microtubule polymerization, arrest mitosis, and induce apoptosis; DNA-damaging agents, such as camptothecin, doxorubicin, and PBD dimers, which insert into DNA double helices causing strand breaks or cross-links, thereby disrupting DNA replication and transcription. The half-maximal inhibitory concentrations (IC₅₀) of these drugs typically range from picomolar to nanomolar levels, sufficient to ensure effective elimination of cancer cells within limited payload capacities.
To overcome the limitations of conventional chemotherapy, researchers are exploring targeted therapeutics as payloads for PDCs. These drugs do not indiscriminately kill all proliferating cells but specifically target cancer gene-dependent signaling pathways. For instance, kinase inhibitors, Bcl-2 inhibitors, and other agents can be conjugated to targeted peptides to precisely interfere with the functions of these key proteins. This strategy holds promise for overcoming resistance issues associated with certain small-molecule targeted drugs, delivering them to tumor cells addicted to specific pathways, enhancing the therapeutic index, and potentially extending applications to non-neoplastic proliferative diseases.
The flexibility of the PDC platform enables it to accommodate more emerging drug delivery categories, demonstrating significant future potential. One such category is protein degradation-targeting chimeras. PROTAC molecules are small molecules that simultaneously bind to target proteins and E3 ubiquitin ligases, inducing the ubiquitination and degradation of target proteins. Combining PROTAC with PDC technology enables tissue-specific degradation of previously "undruggable" targets, making it a hotspot in cutting-edge research. Another category comprises immunomodulators, such as Toll-like receptor agonists or STING agonists. By targeting these immune-stimulating molecules to the tumor microenvironment, "cold" tumors can be transformed into "hot" tumors. This activates the body's innate anti-tumor immune response, enabling synergistic therapy with immune checkpoint inhibitors.
Selecting the optimal linker-payload combination is a complex balancing act. The decision-making process requires comprehensive consideration of multiple factors. First, the biological characteristics of the target must be evaluated. If the target exhibits rapid internalization and abundant lysosomal enzymes, an enzyme-cleavable linker may be preferred. Conversely, if the target is highly expressed only on the tumor cell surface with low internalization efficiency, a linker strategy enabling partial extracellular release should be considered. Second, the payload's mechanism of action is critical. When a potent "bystander effect" is required to eliminate antigen-negative cells within heterogeneous tumors, a cleavable linker should be paired with a payload exhibiting good membrane permeability (e.g., MMAE). Conversely, when strict toxicity control is necessary to avoid damaging normal tissues, a stable, non-cleavable linker combined with a membrane-impermeable payload (e.g., MMAF) offers greater safety. Furthermore, the physicochemical properties of the linker-payload system profoundly influence the drug metabolism and pharmacokinetics of the conjugate. Excessive hydrophobicity can lead to conjugate aggregation and accelerated clearance, typically requiring optimization through the introduction of hydrophilic chains like polyethylene glycol or polarity modifications. Ultimately, only through systematic in vitro and in vivo pharmacodynamic, pharmacokinetic, and toxicological studies can candidate molecules achieving the optimal balance between stability, release efficiency, and safety be selected.
The superiority of theoretical design ultimately requires validation through successful case studies. In the evolution of targeted drug delivery systems, certain classic linker-payload combination strategies have become exemplars within the field, clearly demonstrating how to tailor designs precisely based on target biology, disease characteristics, and drug mechanisms of action. In-depth analysis of these cases provides valuable insights and references for future innovation.
A well-validated successful strategy involves pairing non-cleavable linkers with membrane-impermeable payloads. The core advantage of this approach lies in its exceptional system stability. Because the linker remains exceptionally stable in the bloodstream and the released payload metabolites cannot freely cross cell membranes due to their charged or hydrophilic groups, this combination virtually eliminates "bystander effects." While this may seem to limit its scope, it actually achieves unparalleled precision and safety. This combination is particularly well-suited for disease types where the target antigen is highly and uniformly expressed on tumor cells.
A classic example is a drug conjugate targeting a specific surface antigen on a hematologic malignancy. This drug employs a stable thioether bond as an uncleavable linker, connecting a modified meptol derivative as the payload. Upon release, this derivative exhibits poor membrane permeability due to its chemical structure. Upon binding to target cells and internalization, the conjugate undergoes complete degradation within lysosomes, ultimately releasing a metabolite of the payload still attached to amino acid residues. This design ensures cytotoxic effects are strictly confined to cancer cells expressing the target antigen, minimizing unintended damage to surrounding normal cells—particularly critical hematopoietic stem cells. Clinical studies confirm this strategy demonstrates high response rates and a controlled toxicity profile for treating corresponding indications, establishing it as a model for precision medicine.
In stark contrast to the above strategy, the combination of enzyme-cleavable linkers with highly permeable, potent payloads aims to leverage powerful "bystander effects." This approach targets tumor heterogeneity—a common challenge in solid tumor therapy where not all tumor cells uniformly express target antigens.
A landmark success in this regard is the combination of a cleavable linker based on the valine-citrulline dipeptide sequence with a tubulin inhibitor like monomethyl auristatin E (MMAE). The dipeptide sequence remains stable against various proteases in the bloodstream. However, when the conjugate is internalized by antigen-positive cells and transported to lysosomes rich in cathepsins, the linker is efficiently cleaved, releasing a small-molecule toxin with native activity and hydrophobicity. These toxin molecules freely traverse cell membranes and diffuse into the surrounding tumor microenvironment, effectively killing adjacent antigen-negative tumor cells. This "point-to-area" killing pattern is crucial for eradicating heterogeneous solid tumors. In clinical models of multiple solid tumors, such combinations demonstrated significantly superior efficacy compared to conventional chemotherapy, proving the feasibility of overcoming tumor heterogeneity through the bystander effect.
As research advances, novel cleavable linker strategies combined with diverse payloads continue to expand the application boundaries of targeted drug delivery systems. For instance, a strategy utilizing a β-glucuronidase-cleavable linker paired with a hydrophilic DNA-damaging agent demonstrates unique advantages. This linker exhibits exceptional stability in neutral pH plasma and, due to its hydrophilic nature, reduces the overall hydrophobicity of the conjugate, improving pharmacokinetics. Within the tumor microenvironment, elevated β-glucuronidase activity released from necrotic cells partially cleaves the linker, enabling partial extracellular release. Upon cellular uptake, the conjugate is fully activated under the high enzyme concentrations within lysosomes. This dual-release mechanism, combined with highly potent payloads, demonstrates significant potential for treating solid tumors resistant to conventional therapies.
Another frontier involves pairing non-cytotoxic payloads—such as immunomodulators or proteolysis inducers—with corresponding cleavable linkers. For example, coupling Toll-like receptor agonists to target peptides via phosphodiesterase-sensitive linkers enables tumor-specific activation of immune stimulants, transforming "immunologically cold tumors" into "hot tumors" and opening new avenues for combination immunotherapy. These examples demonstrate that innovative optimization of linkers and payloads is expanding beyond traditional chemotherapy into broader domains of immunotherapy and precision regulation.
Despite significant progress, optimizing linkers and payloads remains fraught with challenges. The foremost challenge lies in tumor heterogeneity and resistance. Existing linker technologies still struggle to overcome resistance caused by downregulated antigen expression or altered internalization pathways. Secondly, limitations in payloads persist, with few potent options available and their inherent toxicity (such as hematotoxicity) remaining dose-limiting factors. Developing novel, highly potent payloads with unique mechanisms of action is an urgent priority. Third, innovations in linker technology require breakthroughs, such as creating "smart" linkers that exhibit hypersensitivity to specific enzymes or reactive oxygen species signals within the tumor microenvironment, enabling more precise controlled release.
Looking ahead, the field will evolve toward diversification and precision. Novel coupling technologies, such as site-specific conjugation, will yield more homogeneous drugs, improving the predictability of pharmacokinetic properties. Bispecific or multispecific PDCs capable of simultaneously targeting multiple antigens hold promise for overcoming tumor heterogeneity. Furthermore, expanding PDC applications beyond oncology into autoimmune diseases, fibrotic disorders, and anti-infective therapies will present novel opportunities and challenges for linker/payload design. Ultimately, rational design leveraging artificial intelligence and computational chemistry will significantly accelerate the translational journey from laboratory to clinical settings, propelling targeted drug delivery systems to new heights.
1. Why is linker and payload selection so important?
The linker and payload define stability, selectivity, and therapeutic performance of a PDC.
2. What types of linkers are most effective?
Cleavable linkers (enzyme-sensitive, pH-sensitive) are favored for tumor targeting, while non-cleavable linkers offer durability.
3. What types of payloads are used in PDCs?
Cytotoxic small molecules, peptides, and innovative payloads like siRNA or immune modulators are commonly employed.
4. What risks exist in poor linker-payload pairing?
Premature release, systemic toxicity, or lack of efficacy are common risks of suboptimal design.
5. How do developers optimize payload selection?
By balancing potency with selectivity, ensuring stable conjugation, and validating in preclinical studies.
6. What innovations are improving linker-payload design?
Bioorthogonal linkers, dual payload strategies, and computational modeling are advancing design precision.
Linker and payload choice defines therapeutic success. We provide customized linker-payload solutions that balance stability, release profile, and efficacy. Our platform helps you overcome development challenges and accelerate PDC programs toward the clinic.
