Design and Bioanalysis of Peptide-Drug Conjugate (PDC)

Design and Bioanalysis of Peptide-Drug Conjugate (PDC)

Peptide-Drug Conjugates (PDCs) represent the next generation of targeted therapeutic agents following Antibody-Drug Conjugates (ADCs). The core advantages of PDCs include enhanced cellular permeability and increased drug selectivity. PDCs are primarily composed of three parts—peptides, linkers, and cytotoxic payloads.

Compared to non-targeted anticancer drugs, PDCs significantly extend blood circulation time, increase maximum tolerated doses, enhance tumor accumulation, and improve anticancer bioactivity. Since normal cells lacking target receptors do not bind with tumor-targeting peptides, compounds accumulate in receptor-positive tumor cells, allowing for reduced drug dosages and minimized side effects. Additionally, PDCs can leverage peptide properties to improve drug solubility, permeability, and selectivity.

In comparison to ADC drugs, PDC drugs offer several advantages: smaller molecular weight, strong tumor penetration, effective inhibition of solid tumors, and ease of synthesis of single homogeneous substances due to their small size. PDCs also exhibit low immunogenicity and a broad range of cytotoxic drug options. Due to their strong tumor tissue penetration, PDCs can accumulate at the target site, allowing for the use of chemotherapy drugs like doxorubicin and paclitaxel, which have relatively lower toxicity and widespread clinical application. Additionally, PDCs have a simple production process and are easily scalable, resulting in lower production costs.

Mechanism of Action of Peptide-Drug Conjugates

The mechanism of action of PDCs is similar to ADCs, utilizing linkers to covalently connect targeted peptides and cytotoxins. This precise targeting of specific receptors on tumor cells allows controlled release of cytotoxins, leading to the destruction of tumor cells. In early development, besides studying the overall pharmacological/therapeutic effects of PDCs, research is also needed on the pharmacological effects of each individual component.

PDC Design

Targeting peptides directly deliver drugs to target cells, restricting the off-target effects of chemotherapy drugs. Various techniques can be used to determine their target binding affinity, including surface-enhanced raman scattering (SERS), bio-layer interferometry (BLI), isothermal titration calorimetry (ITC), and drug affinity response target stability (DARTS). The secondary structure of targeting peptides significantly affects their binding affinity. Therefore, it is crucial for linkers to stabilize the secondary structure to enhance the binding affinity of targeting peptides. When linkers bind with chemotherapy drugs, the secondary structure of targeting peptides must be maintained.

The special structure and mechanism of action of PDCs increase uncertainty in their stability in the matrix. Stability issues can be addressed by considering the stability of peptides, linkers, and cytotoxic payloads separately, and by adding enzyme inhibitors, adjusting pH values, adding reducing agents, and controlling experimental environments.

Optimization of peptides in PDCs. Fig. 1 Optimization of peptides in PDCs. (Bethany, 2021)

  • Amino Acid Modification

One method to increase peptide stability is to use D-amino acids instead of L-amino acids. For example, the substitution of D-amino acids for two amino acid sites in the Octreotide increases its half-life from a few minutes to 1.5 hours, resulting in improved PK characteristics.

Polyethylene glycol (PEG) is a candidate material for peptide modification due to its low cost, high bioavailability, biocompatibility, and non-immunogenicity. In the case of the short half-life peptide HM-3, mPEG-ALD is the preferred N-terminal linker for PEG modification, extending the half-life of HM-3 peptide by 5.86 times.

  • Peptide Stapling (PS)

Peptide stapling has two subtypes: single-component PS (1C-PS) and dual-component PS (2C-PS). In 1C-PS, intramolecular bonds between non-natural amino acid side chains can be cyclized, typically depending on higher-order structures. The example of 1C-PS utilized ring-closing metathesis (RCM) with O-allylserine residues.

Peptides in PDCs

Peptides, as crucial components of PDCs, form the foundation for achieving various functions. Connecting water-soluble peptides can enhance the solubility of hydrophobic drugs. Peptides can be classified based on their functions into cell-penetrating peptides (CPP), cell-targeting peptides (CTP), and responsive peptides. CPP refers to small molecules or short-peptide sequences with 5-30 amino acids that can enter cells without disrupting membrane integrity. CPPs exhibit positive charge and amphiphilic characteristics in physiological environments and have found widespread applications in drug delivery systems.

CTPs include passive targeting and active targeting. Passive targeting involves the passive accumulation of drugs in diseased tissues due to the nature of the delivery system or the characteristics of the target tissue. Active targeting, on the other hand, delivers drugs to diseased tissues by recognizing receptors or proteins specifically expressed in the target tissue. Most PDCs based on CTPs are delivered through active targeting mechanisms. Responsive peptides undergo structural changes under external stimuli, such as temperature, pH, enzymes, etc. The weak acidity of the tumor microenvironment has also become a new pathway for targeting tumors.

Linkers in PDCs

Linkers determine the circulation time and stability of PDCs in the body. Ideal linkers should remain stable during circulation to avoid premature drug release. Once reaching the diseased tissue, they should release the drug rapidly and effectively. Additionally, linkers should not affect the affinity of peptides to their receptors and the activity of drugs. Furthermore, the synthesis process of linkers carrying peptides and drugs should be as simple as possible while maintaining stability throughout the entire synthesis process. The hydrophobicity of linkers should not be too strong to prevent aggregation of PDCs, leading to poor in vivo stability, reduced efficacy, and strong systemic toxicity and immune side effects. According to the drug release mechanism and the cleavage behavior of linkers, linkers are mainly classified into non-cleavable linkers and cleavable linkers. Cleavable linkers are the most commonly used linkers in PDC construction. They can cleave and release drugs in physiological environments or in the presence of enzymes, including protease-sensitive, pH-sensitive, and redox-sensitive types.

Payloads in PDCs

The cytotoxic drugs often have disadvantages such as low water solubility, poor selectivity, short half-life, and poor stability, limiting their clinical application. Drugs delivered through the PDC strategy need feasible binding sites. Additionally, drugs should not have pharmacological activity in the bound form. When released in diseased tissues, they should continue to exhibit a clear mechanism of action and strong pharmacological activity. Peptide conjugation enhances drug solubility, promotes drug selectivity, prolongs in vivo circulation time, optimizes bioavailability, and prevents side effects and toxicity to other tissues.

Bioanalysis of PDCs

The coupled structural characteristics of PDCs make their in vivo processes diverse, resulting in the complexity of bioanalytical methods. Pharmacokinetics (PK) and toxicokinetics (TK) studies mainly detect PDCs, peptides, and free small molecules. The toxicity of PDCs is closely related to the characteristics of peptides, small molecules, and linkers, and as these components change, the toxicity response characteristics also change. In non-clinical safety studies, attention should be paid to the impact of drug composition structure and pharmacokinetic characteristics on toxicity, and experimental results should be comprehensively analyzed. In LC-MS/MS, PDCs generate multi-charged ions with different m/z values. Sensitivity can be adjusted to meet analysis needs by changing the charge distribution, optimizing instrument parameters, and liquid phase conditions, among others. Ensuring the extraction recovery of PDCs in the matrix is crucial, and care should be taken to avoid structural damage during pre-processing.


  1. Bethany M., et al. Peptides as a platform for targeted therapeutics for cancer: peptide–drug conjugates (PDCs), Chem. Soc. Rev., 2021, 50, 1480-1494.
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