Peptide-Drug Conjugates (PDCs): Design, Stability, and Future Prospects
Online Inquiry
Peptides are compounds composed of two or more amino acids linked by peptide bonds. Typically consisting of 10 to 100 amino acids, peptides have a molecular weight ranging from approximately 500 to 10,000 Daltons. Their size places them between small-molecule chemical drugs and large-molecule protein therapeutics, making them a unique class of "proteins." Due to their relatively small molecular weight, peptides can be directly absorbed by the human body. Peptides are widely present in biological systems, with tens of thousands of types discovered. They play crucial roles in regulating and controlling the functions of various systems, organs, tissues, and cells, making them essential for life processes.
Peptide-based drugs are peptides with specific therapeutic effects obtained through chemical synthesis, genetic recombination, or extraction from animals and plants. These drugs typically consist of 10 to 50 amino acids, with molecular weights generally exceeding 1,000 Daltons (Da) but remaining below 10,000 Da. Peptide drugs combine the advantages of both large and small molecules—they are relatively easy to synthesize, modify, and optimize. Compared to monoclonal antibody drugs and recombinant protein therapeutics, peptide drugs exhibit higher activity, are easier to isolate from impurities, and achieve high purity. Their relatively simple spatial structure also contributes to their stability. Additionally, peptide drugs generally have low immunogenicity or even no immunogenicity. Compared to small-molecule chemical drugs, peptides also share the specificity and strong therapeutic efficacy of protein-based drugs. In terms of production, peptide drugs share similarities with small-molecule drugs, including controllable quality, well-defined structures, and relatively low production costs.
Based on their properties and applications, peptide drugs can be classified into four main categories:
Limitations and Optimization Strategies for Peptide Drugs
Despite their advantages, peptide drugs have several limitations, including enzymatic degradation at terminal sites, chemical instability, and rapid renal clearance, all of which hinder in vivo studies and reduce bioavailability. Various strategies have been developed to improve the ADME (Absorption, Distribution, Metabolism, and Excretion) properties of peptides, such as cyclization modifications, amino acid modifications, conjugation with macromolecules, and formulation optimization. These approaches have been proven to enhance PDC pharmacokinetic (PK) properties by improving cellular permeability, increasing chemical and proteolytic stability, and reducing overall renal clearance, thereby extending circulation half-life.
Cyclization reactions are widely used in peptide synthesis, including head-to-tail cyclization, head/tail-to-side-chain cyclization, and side-chain-to-side-chain cyclization. Bicycle Therapeutics, as indicated by its name, designs peptide-drug conjugates (PDCs) with a bicyclic peptide structure that binds to target proteins with high affinity and selectivity, similar to antibodies.
Amino Acid Modifications
Amino acid side chains provide another effective avenue for peptide modification. By disrupting enzymatic recognition sites and increasing steric hindrance, side-chain modifications can enhance peptide stability. One common strategy is substituting L-amino acids with D-amino acids. For example, octreotide, a peptide drug used to treat gastrointestinal tumors, incorporates D-amino acid substitutions at two sites, increasing its half-life from just a few minutes to 1.5 hours and significantly improving its PK properties.
Polyethylene glycol (PEG) is a widely used candidate for peptide modification due to its affordability, high bioavailability, strong biocompatibility, and non-immunogenicity. For instance, HM-3, a peptide with a short half-life requiring twice-daily administration, needed an extended half-life to improve intracellular drug retention. The preferred PEG modification linker, mPEG-Ald (methoxy-polyethylene glycol-aldehyde), was applied at the N-terminus, extending HM-3's half-life by 5.86 times.
Other commonly used macromolecules for peptide modification include polysialic acid (PSA) and hydroxyethyl starch (HES).
Formulation Optimization
Intracellular protein delivery systems often rely on genetic fusion of proteins with membrane-penetrating tags or encapsulation using cationic liposomes, polymers, or inorganic nanomaterials. Several formulation-based strategies, such as permeability enhancers and acid-stable coatings, have been reported to improve the oral bioavailability of peptide therapeutics. Permeability enhancers facilitate peptide transport across epithelial cells by disrupting tight junctions and adhesion proteins. Another strategy to enhance oral drug absorption is the use of acid-resistant coatings, which remain intact in the low pH environment of the stomach but dissolve as pH increases in the colon and rectum, leading to drug release at the intended site.
Peptide-Drug Conjugates (PDCs)
Peptide-drug conjugates (PDCs) represent an emerging targeted therapeutic approach that enhances tumor penetration and selectivity. PDCs primarily consist of three components: a peptide, a linker, and a cytotoxic payload.
Although PDCs share conceptual similarities with antibody-drug conjugates (ADCs), their structures and properties are significantly different. ADCs typically have a molecular weight exceeding 150 kDa, whereas PDCs generally weigh only a few kDa. Compared to ADCs, PDCs exhibit improved tumor penetration and minimal immunogenicity. Additionally, their metabolic pathways differ: PDCs are primarily metabolized through the kidneys, while ADCs are processed via the liver. Due to the shorter peptide sequences in PDCs, their structures are more flexible, making it easier to introduce non-natural amino acids, form cyclic peptides, and incorporate various modifications and conjugations. These enhancements improve PDCs' targeting ability and stability.
Peptide Modification Services at Creative Peptides
FDA Approved PDC Drugs
Currently, several PDC drugs have been approved worldwide, though most are used for diagnostic purposes, with only three approved for therapeutic applications.
Diagnostic PDCs
Octreoscan® (111In-DTPA-Octreotide) was the first approved PDC drug, authorized for diagnostic imaging of SSTR-positive tumors. It enables tumor localization through intravenous injection. Following this, multiple PDCs for diagnostic imaging have been approved, including NETSPOT® (68Ga-DOTATATE) and TOCscan® (68Ga-DOTATOC).
Therapeutic PDCs
Lutathera was the first PDC approved for therapeutic use and is categorized as a peptide receptor radionuclide therapy (RDC). It targets somatostatin receptors (SSTR), enters cells, and releases the radionuclide 177Lu, which emits radiation to damage tumor cells. In 2018, the FDA approved Lutathera for treating gastroenteropancreatic neuroendocrine tumors (GEP-NETs).
Pepaxto (developed by Oncopeptides) was the first PDC targeting aminopeptidases. Approved by the FDA in February 2021, it was a covalent conjugate of a DNA alkylating agent and a peptide targeting aminopeptidases. Due to its high lipophilicity, it entered cells and was hydrolyzed by aminopeptidases, releasing a hydrophilic DNA alkylating agent that induced DNA damage and tumor cell death. Pepaxto was approved for use in combination with dexamethasone for relapsed/refractory multiple myeloma (RRMM). However, in October 2021, the drug was withdrawn from the U.S. market following a Phase III clinical trial failure.
Pluvicto (developed by Novartis) represents another milestone in radiopharmaceutical therapy. Approved in 2022, it is an RDC targeting prostate-specific membrane antigen (PSMA) for the treatment of prostate cancer. Pluvicto (177Lu-PSMA-617) consists of the radionuclide 177Lu, a chelator (DOTA), a linker, and a targeting structure.
An Important Component of PDC-Peptides
As a crucial component of peptide-drug conjugates (PDCs), targeted peptides within PDCs are classified into two main categories: cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs). Both CPPs and CTPs facilitate the targeted delivery of cytotoxic agents to diseased tissues, increasing their local concentration while minimizing toxic effects on non-diseased tissues. This reduces adverse reactions and ultimately enhances therapeutic efficacy while lowering toxicity.
Cell-Penetrating Peptides (CPPs)
Commonly used CPPs include Pep-1, Penetratin, PepFact14, and Transportan. CPPs are short peptides (fewer than 30 residues) capable of transmembrane transport and can be classified into three categories: protein-derived CPPs, modified CPPs, and designed CPPs. The exact mechanism by which CPPs are taken up by the cell membrane is not yet fully understood. Some CPPs traverse the membrane through energy-dependent cellular processes (such as endocytosis or receptor-mediated uptake), while others utilize energy-independent, non-endocytic translocation pathways. However, due to their low cell specificity, the application of CPPs is somewhat limited.
Cell-Targeting Peptides (CTPs)
The first point of interaction between a drug or delivery carrier and a cell is typically the cell membrane. Thus, receptors that are abnormally expressed on tumor cells can serve as targets for directed delivery. CTPs generally consist of 3 to 14 amino acids. Common CTPs include PEGA (a peptide molecule that binds to tumor vascular surface molecules), somatostatin analogs, bombesin analogs, and RGD (Arg-Gly-Asp cyclic peptides). CTPs interact with receptors that are overexpressed on specific cells. Once conjugated with a therapeutic drug, CTPs facilitate drug transport and accumulation at the target site, significantly reducing drug-related side effects.
An Important Component of PDC-Linkers
Linkers serve as a bridge connecting tumor-homing peptides with cytotoxic molecules, playing a vital role in maintaining the structural integrity of PDCs and controlling the release of toxic molecules. Based on the mechanism of action of PDCs, an ideal linker should possess the following characteristics:
- An appropriate length;
- No interference with the binding ability of the homing peptide to its receptor;
- Sufficient stability to prevent premature or nonspecific drug release outside the target site, thereby reducing adverse effects;
- Rapid cleavage upon reaching the tumor target site, ensuring the timely release of the cytotoxic agent;
- Moderate hydrophobicity, as excessive hydrophobicity may lead to PDC aggregation, reducing in vivo stability and efficacy while also causing severe systemic toxicity and immune responses.
Classification of Linker
The functional groups commonly found in linkers can be categorized into four types: non-cleavable linkers, enzyme-cleavable linkers, acid-cleavable linkers, and redox-sensitive linkers.
Non-Cleavable Linkers
Non-cleavable linkers represent the simplest form of conjugation. Common non-cleavable linkers include esters, amides, thioethers, oxime bonds, triazoles, and γ-aminobutyric acid. These linkers remain stable in the bloodstream, preventing premature drug release before reaching the tumor tissue and thereby reducing off-target toxicity.
Enzyme-Cleavable Linkers
Enzyme-sensitive linkers are the most widely used in PDC drugs. Short peptides with specific sequences are common enzyme-sensitive linkers that can be cleaved by specific proteases. These proteases are usually inactive in the extracellular environment due to pH conditions and serum protease inhibitors, ensuring that enzyme-sensitive linkers remain stable in plasma. However, they can be selectively recognized and cleaved by proteases overexpressed in tumor cells. Tumor tissues often exhibit high levels of proteases such as cathepsin B (CatB) and matrix metalloproteinases (MMPs). For instance, CatB can enzymatically degrade short peptide linkers like Val-Cit, Val-Ala, and Gly-Phe-Leu-Gly.
Acid-Cleavable Linkers
Tumor cells exhibit rapid metabolic activity and tend to ferment glucose into lactic acid even in the presence of oxygen, leading to a lower pH (6.2–6.8) in tumor tissues compared to the physiological pH (7.4). Research has shown that lysosomal pH is even lower (4.5–6.0), creating a significant pH difference that enables acid-sensitive linkers to remain stable in the circulatory system but rapidly cleave upon reaching the tumor microenvironment or intracellular compartments, releasing the toxic payload. Currently, the most widely used acid-sensitive linker is the hydrazone bond, which is stable under neutral conditions but readily hydrolyzed in mildly acidic environments.
Redox-Sensitive Linkers
Redox-sensitive linkers in PDCs typically contain disulfide bonds, which can be cleaved by reduced glutathione (GSH). GSH plays a crucial role in cellular redox balance and is present at a concentration 1000 times higher in cells than in plasma. Moreover, the concentration of GSH in tumor cells is four times higher than in surrounding normal cells. This disparity ensures that PDCs containing disulfide bonds remain relatively stable in plasma but selectively release their cytotoxic payload within tumor tissues.
An Important Component of PDC-Payload
Many cytotoxic drugs exhibit strong pharmacological activity but suffer from poor solubility, lack of selectivity, and short half-lives, leading to significant side effects and multidrug resistance that limit their therapeutic applications. By conjugating cytotoxic molecules with tumor-homing peptides, these limitations can be mitigated, improving pharmacokinetic properties and expanding the therapeutic window.
Cytotoxic molecules used in PDC drugs typically need to meet the following criteria:
- The presence of functional groups that allow conjugation with peptides and linkers;
- High cytotoxicity against tumor cells.
Common cytotoxic molecules used in PDCs include doxorubicin (Dox), paclitaxel (PTX), camptothecin (CPT), docetaxel (DOC), and monomethyl auristatin E (MMAE). In addition to cytotoxic agents, PDC payloads may also include radionuclides and oligonucleotides.
Doxorubicin (Dox)
DOX is a well-known Topo II inhibitor approved by the FDA for cancer treatment, demonstrating high activity against various cancers such as lymphoma, soft tissue sarcoma, breast cancer, and genitourinary cancers. However, it often causes adverse effects, including cardiotoxicity, myelosuppression, and nausea. Moreover, DOX readily induces multidrug resistance, limiting its clinical applications. Conjugating DOX with peptides can enhance its targeting capability, efficacy, pharmacokinetics, and reduce adverse effects.
Camptothecin (CPT) and Its Derivatives
CPT and its derivative, SN-38, are TOPO I inhibitors that can directly disrupt DNA structure and inhibit DNA topoisomerase I, preventing DNA strand re-ligation and thereby blocking DNA replication and RNA synthesis. CPT is a cell cycle-specific drug that mainly acts on the S phase. However, its poor water solubility, instability of the lactone ring at physiological pH, severe adverse effects, low delivery efficiency, and high off-target toxicity limit its clinical applications. Studies have shown that constructing peptide-drug conjugates (PDCs) based on CPT and SN-38 can effectively improve their pharmacological properties.
Microtubule Polymerization Inhibitors
Microtubule polymerization inhibitors such as paclitaxel (PTX), docetaxel (DOC), maytansinoid (DM1), and monomethyl auristatin E (MMAE) exhibit strong anticancer activity. However, side effects such as neurotoxicity and poor water solubility hinder their further development. Conjugating these microtubule inhibitors with peptides can overcome these limitations.
DNA Synthesis Inhibitors
Chlorambucil (CLB) and gemcitabine (GEM) are DNA synthesis inhibitors with potent anticancer activity. CLB is an alkylating agent capable of inserting alkyl groups into double-stranded DNA, while GEM is a nucleoside analog that incorporates into DNA and disrupts its synthesis. Both CLB and GEM have been widely used in cancer treatment. Unfortunately, their clinical development is severely hindered due to significant side effects and drug resistance. Conjugating CLB and GEM with peptides is an effective strategy to address these drawbacks.
Tyrosine Kinase Inhibitors (TKIs)
Tyrosine kinase inhibitors (TKIs) are widely used as anticancer drugs that block abnormal cell growth and proliferation by inhibiting tyrosine kinase-regulated signaling pathways. For example:
- Crizotinib is a potent multi-target kinase inhibitor targeting ROS1, anaplastic lymphoma kinase (ALK), and c-Met kinase, approved for cancer treatment.
- Gefitinib is an epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor.
- Sunitinib is a promising anti-angiogenic agent that inhibits multiple receptor tyrosine kinases (RTKs), including VEGFR-1/2, PDGFR-α/β, KIT, and FLT3.
However, the off-target effects caused by the expression of target kinases in normal tissues hinder the development of TKIs. Conjugating TKIs with peptides may enhance selectivity, reduce toxicity, and improve efficacy.
Radionuclides
PDCs are not limited to therapeutic applications but are also widely used as imaging agents. The first FDA-approved radiolabeled peptide-based PDC, 111In-DTPA-octreotide (Octreoscan), was approved for diagnostic imaging of SSTR-positive tumors, allowing tumor localization via intravenous injection. Since then, several other PDCs have been approved for diagnostic imaging, such as NETSPOT® (68Ga-DOTATATE) and TOCscan® (68Ga-DOTATOC).
Lutathera was the first PDC drug approved for therapeutic use globally. It is a peptide receptor radionuclide therapy (RDC) targeting somatostatin receptors, entering tumor cells and releasing the radionuclide 177Lu, which emits radiation to damage tumor cells. In 2018, the FDA approved Lutathera for treating gastroenteropancreatic neuroendocrine tumors (GEP-NETs).
In 2022, Novartis' Pluvicto, another RDC drug targeting PSMA, was approved for treating prostate cancer, marking a milestone in radiopharmaceutical therapy. Pluvicto (177Lu-PSMA-617) consists of the radionuclide 177Lu, a chelator (DOTA), a linker, and a targeting structure.
Other Payloads
In addition to cytotoxic and targeting payloads, various other payloads have been applied in PDC design, such as siRNA and antisense oligonucleotides (AONs).
siRNA (Short Interfering RNA)
siRNA consists of 20–25 base-pair double-stranded RNA molecules. Once inside the cell, siRNA is incorporated into the RNA-induced silencing complex (RISC), which contains the endoribonuclease Ago2 to cleave target mRNA. Both CPPs (cell-penetrating peptides) and CTPs (cell-targeting peptides) have been conjugated with siRNA to enhance its delivery and targeting.
Chiu et al. conjugated TAT 47–57 with CDK9-targeting siRNA to form TAT-siRNA conjugates. Immunohistochemistry showed that TAT-siRNA treatment led to the specific knockdown of SOD1, Casp3, and Casp9.
Shuai H et al. added a cRGD peptide to methoxy-modified EGFR siRNA, forming cRGD-EGFR siRNA, which significantly inhibited tumor growth, reduced EGFR expression, and downregulated EGFR mRNA and protein levels in tumor tissues.
Antisense Oligonucleotides (AONs)
AONs are single-stranded DNA or RNA sequences complementary to a selected target sequence. They bind to the messenger RNA (mRNA) of the target gene, silencing its expression. AONs have been widely used to interfere with biological processes and, in some cases, have been developed as therapeutic drugs.
Susan M et al. reported that phosphorodiamidate morpholino oligomer (Pip) efficiently delivers single-stranded antisense splice-switching oligonucleotides (SSO). In a mouse model, Pip delivered SSOs at a significantly lower dose than naked SSOs, effectively alleviating spinal muscular atrophy (SMA).
Key Considerations in PDC Molecular Design
When conjugating a cytotoxic payload to a peptide, the modification site of both the cytotoxic payload and the peptide is a primary consideration. A thorough understanding of the structure-activity relationship of the peptide and the payload is necessary when selecting the conjugation site to avoid compromising their activity.
Drug Conjugation Site
Selecting an appropriate conjugation site during modification is crucial to prevent the loss of drug activity. The conjugation site should be positioned away from functional groups. For example, methotrexate is an antifolate agent that primarily inhibits dihydrofolate reductase (DHFR) to block tumor cell growth and proliferation. Methotrexate has a molecular structure similar to folic acid, allowing it to compete with folic acid for DHFR binding, thereby reducing folic acid binding and inhibiting tetrahydrofolate synthesis. The pteridine moiety of methotrexate is essential for its antimetabolic activity, and any modification to this region reduces drug potency. In contrast, modifications to the glutamate residue have minimal impact on DHFR activity, making it an ideal conjugation site.
Peptide Conjugation Site
After conjugation with a cytotoxic drug, the spatial structure and hydrophobicity of the peptide may be altered, potentially affecting its cellular penetration or targeting efficiency. The relationship between peptide structure and function is a fundamental criterion for selecting the conjugation site-critical functional sites should not serve as conjugation sites.
Amino acids such as lysine, cysteine, glutamate, and serine, which do not participate in receptor recognition, can be directly used for payload conjugation through their side chains. Alternatively, non-essential residues can be replaced with these amino acids to introduce potential modification sites. Many peptides also permit simple N-terminal modifications since their N-terminal regions do not participate in receptor recognition. Ideally, the peptide carrier should include multiple modification sites, enabling each peptide molecule to incorporate multiple payloads, thereby increasing the drug concentration at the tumor site and enhancing therapeutic efficacy.
Conclusion and Future Outlook
Traditional chemotherapy is increasingly inadequate due to severe side effects, making it difficult to meet patient needs. PDCs, as a precise tumor-targeted therapeutic approach, expand the therapeutic window, enhance treatment efficacy, and shift the paradigm of tumor drug therapy compared to conventional chemotherapeutics. Several PDCs have already been successfully applied in clinical settings, demonstrating their vast potential and high development value. Consequently, extensive research efforts focus on the development of novel PDCs. However, challenges such as poor stability and short half-life persist. While no perfect solution exists yet, advancements in the field suggest that PDCs will soon find broader clinical applications. The field of PDC-targeted drug delivery systems holds unlimited potential and will require further research in the coming years. Future trends and research hotspots may include the following:
1. Development of PDCs Based on Humanized Antibodies
Since the introduction of monoclonal antibodies (mAbs), murine-derived mAbs have played a significant role in clinical diagnosis and therapy. However, murine mAbs exhibit strong immunogenicity, making the development of PDCs based on humanized antibodies crucial for ensuring safety and efficacy.
2. Enhancing the Biological Stability of PDCs
Compared to small-molecule drugs, targeted PDCs exhibit high specificity but suffer from poor biological stability. To improve in vivo stability and ensure efficacy, modifications of targeting peptides and linkers are under investigation. Various nanodrug delivery systems with specific functions have been successfully developed and widely applied in cancer therapy. Integrating PDCs with nanodrug delivery systems to develop novel multifunctional antibody-targeted or peptide-targeted nanodrug delivery platforms is a promising strategy for overcoming PDC instability.
Computationally Assisted PDC Design
Unlike ADCs, which primarily rely on biological and chemical technologies, PDCs can significantly benefit from computational drug design methods. Computational analysis allows for a deeper understanding of interactions between targeting peptides and their ligands. To optimize peptide design and subsequent PDC development, docking methods, molecular dynamics simulations, and artificial intelligence-driven algorithms can predict, calculate, and evaluate the binding affinities between receptors and ligands. Additionally, computational approaches can be used to design novel peptides for target proteins or predict potential target proteins and binding sites for specific peptides.
PDCs Targeting Multiple Receptors or Antigens (Analogous to Bispecific ADCs)
Currently, most marketed or investigational PDCs rely on peptides targeting a single receptor. If the targeting peptide can bind to different sites on the same antigen or multiple overexpressed receptors on the same cancer cell surface, it could enhance receptor clustering and accelerate the internalization of conjugated drugs.
Design of Dual-Payload PDCs (Analogous to Dual-Drug ADCs)
Classical PDCs consist of a peptide, linker, and drug payload. However, drug resistance is an inevitable challenge in single-agent PDC therapy. With rapid advancements in drug formulation technologies, dual-payload PDCs incorporating two cytotoxic agents with distinct mechanisms of action offer a promising strategy to overcome drug resistance associated with monotherapy.
In conclusion, as research on PDC-targeted drug delivery systems advances, the development and application of highly selective, biocompatible, and safe PDCs will expand significantly, contributing to sustainable human health development.
Copyright © 2025 Creative Peptides. All rights reserved.
