Peptides play multiple functions in the process of human life, such as repairing cells, improving cell metabolism, preventing cell degeneration, etc. Peptides are biologically active and have excellent targeted transport capabilities. This capability is applicable not only to oncology, but also to targeted therapies for COVID-19, diabetes, rheumatism, and rheumatoid arthritis.
Peptide-drug conjugates (PDCs) are the next generation of targeted therapeutics after ADCs, and their core advantages are enhanced cell permeability and improved drug selectivity. Peptide-drug conjugates (PDCs) of comparable potency have broader applications than current antibody-drug conjugates (ADCs). In recent years, pharmaceutical companies have been developing PDCs as targeted therapeutic candidates for cancer, COVID-19, metabolic diseases, etc.
Targeted Therapy Strategies
Cancer is the second leading cause of death worldwide. Depending on the patient's stage and tumor type, the patient is given one or more of the following treatments: surgery, radiation, or chemotherapy. Drug therapy has different levels of toxicity and side effects, and some serious side effects are the direct cause of limiting drug dosage or use. In cancer therapy, three targeted therapy modalities are used to enhance the nonspecific and antitumor activity of cancer therapy. First, targeted therapeutic drugs can inhibit the expression of the protein, such as protein kinases or enzymes. Another approach is to fuse an effective molecular structure (such as ADC, toxin small molecule or CAR-T) to an overexpressed protein on the surface of tumor cells and synergistically inhibit tumor cell division while delivering a cytotoxic payload or stimulating a tumor-directed immune response. The third approach is the application of PDC, which drives the accumulation of toxic payloads in tumor cells.
The biggest challenge for transporting proteins and peptides in the body is their instability. Consideration should be given to molecular size, molecular charge, protein internal structure, solvent effects, lipid membrane accumulation and hydration, stability, affinity for receptors, etc. In ADCs, specific monoclonal antibodies (mAbs) that express antigens on cancer cells can be used to transport cytotoxic substances while reducing damage to normal cells, resulting in better therapeutic outcomes and enhanced drug metabolism. However, mAb can cause immunogenicity. PDCs and ADCs are similar in concept but have distinct structures and characteristics. Antibodies have higher specificity and longer half-life, while targeting peptides have better drug-loading ability and tissue penetration ability. In addition, multipurpose linear or cyclic peptides are more prone to structural changes. The comparison between PDCs and ADCs is shown in Table 1.
Table 1 The comparison of PDC and ADC
General Considerations For PDC And Its Clinical Trials
Peptide-drug conjugate (PDC) is a kind of targeted therapy drug, its function is similar to that of ADC. PDC used for tumor therapy generally consists of three components: homing peptide, linker and payload. All three components coordinate the delivery of chemotherapy drugs by targeting receptors on tumor cells to amplify their therapeutic effects.
Figure 1. Structure diagram of peptide-drug conjugate
(Image source: References )
Homing peptides are an important component of PDC. Bicyclic toxin peptides (BTPs), dendritic peptides, and self-assembly enhancing peptides have been demonstrated as drug delivery systems. PDCs have several advantages such as deeper tumor penetration, less immunogenicity and faster renal clearance. The drug is linked to BTP to ensure conformational stability. In 2021, Bicycle Therapeutics (Nasdaq: BCYC) announced three experimental BTC (Bicycle Toxin Conjugate) drugs: BT1718, BT5528 and BT8009. All patients are in Phase I/II clinical development. BT1718 is a novel bicyclic peptide anticancer drug that targets membrane type I matrix metalloproteinases to release its toxic payload, DM1. BT5528 showed preliminary antitumor activity as a drug targeting EphA239. BT8009 is a BTC targeting Nectin-4, which has shown excellent anti-tumor activity in preclinical studies. Nectin-4 is a type I membrane protein that is overexpressed in most tumors, including urothelial, breast, pancreatic and triple-negative breast cancer (TNBC), and can affect cell proliferation, differentiation, migration and invasion.
In clinical trials of PDCs, two therapeutic PDCs are currently approved on the market: 177Lu-dotatate(lutathera) and melflufen. The first PDC drug 177Lu-dotatate approved by the U.S. Food and Drug Administration (FDA) is for the treatment of gastroenteropancreatic neuroendocrine tumors (GEP-NETs). Melflufen is indicated for the early treatment of refractory multiple myeloma (MM) by rapidly releasing the alkylating agent into the tumor via aminopeptidase in the cell. However, in October 2021, the company oncopeptides AB announced its decision to withdraw melflufen from the U.S. market due to the failure of a Phase III clinical trial to successfully reduce the risk of death in patients with relapsed refractory multiple myeloma (HR=1.104).
Table 2 PDC in clinical trials
Homing Peptide in PDC
The selection of peptides affects the efficiency of drug endocytosis in PDCs. Once the target has been selected, it is also important to select the appropriate peptide for PDC, which has significant effects on efficacy, pharmacokinetic/pharmacodynamic characteristics, and therapeutic indices. The ideal peptides of PDCs should have strong target-binding affinity, high stability, low immunogenicity, high efficiency internalization and long plasma half-life.
Homing peptides are specifically targeted overexpressed protein receptors in tumor tissues. It directly delivers the loaded drug to target cells, limiting off-target delivery of chemotherapy drugs. These homing peptides have been previously reported to have high binding affinity to target sites at nanomolar concentrations. To determine their target-binding affinity, a variety of techniques can be used, including surface-enhanced raman scattering (SERS), bio-layer interferometry (BLI), isothermal titration calorimetry (ITC), and drug affinity responsive target stability (DARTS).
Figure 2. Homing Peptide and Target Binding Affinity Assay Technique
(Image source: References )
These peptides are also hydrophobic, amphiphilic, and favorable for negative charges across cell membranes. Cell-penetrating peptides not only deliver the drug to the target tissue, but also allow for cellular internalization. Positively charged CPPs have some disadvantages, such as unstable target selectivity, leading to non-specific cellular uptake. Therefore, anion CPPs are often used in PDC to improve tumor cell specificity.
Peptides are ideal carrier molecules because they have the same capabilities as monoclonal antibodies. They have a high affinity for overexpressed receptors on the surface of tumor cells, without the drawbacks of mAb. However, the binding of the payload to the peptide molecule is particularly critical because the spatial structure of the payload affects receptor binding and selectivity, thus interfering with receptor recognition. Therefore, understanding peptide receptor interactions is essential for rational drug selection.
Pharmacokinetics of Peptides
When designing drugs, it is important to study pharmacokinetics (PK) and pharmacodynamics (PD). Peptides and small molecules have different pharmacokinetic characteristics. Like ADCs, peptides are not easy to take orally. However, oral administration is one of the most convenient methods and patients have better medication compliance.
Figure 3 Peptide properties in vivo and in vitro
(Image source: References )
The half-life and in vivo circulation time of peptides are shorter than those of biomacromolecules, and the frequency of administration is slightly higher, resulting in slow research on ADCs and PDCs. The half-life of hydrophilic peptides is determined by several soluble enzymes in blood and cell membranes. Exopeptidase is one of the most important soluble enzymes. This is related to the catabolism of the peptide and the chemical instability of the plasma.
Rapid renal clearance and short half-life hinder the research of peptides in vivo and affect their druggability. The FDA recently approved the oral peptide N-[8-(2-hydroxybenzyl)alanine] (SNAC) as an adjunct to albiglutide for the treatment of type 2 diabetes. SNAC acts as a buffer in the stomach and reduces the activity of proteolytic enzymes. Although semaglutide has been approved for use by the FDA, oral peptides still have a long way to go before clinical application.
There are several ways to improve the ADME properties of peptides, such as increasing cell permeability, enhancing chemical stability, anti-proteolytic ability, and reducing renal clearance, thereby prolonging the in vivo circulatory half-life. Prolonging half-life is beneficial for economic justification and patient compliance. Based on this characteristic, PDC administration can have a wide range of dose adjustment.
Linkers in PDC
The selection of linkers is one of the key factors in the design of PDC, and the microenvironment of PDCs needs to be considered so as not to interfere with the binding affinity of peptides to their receptors and drug efficacy. The types of linkers used in PDCs vary, depending on their length, stability, release mechanism, functional group, hydrophilicity/hydrophobicity, and other characteristics. Linkers used in PDCs must exhibit stability to prevent premature and nonspecific drug release.
The linker needs to have a certain level of stability, so that it can ensure the integrity of the circulation process before the PDC reaches the tumor cells, avoiding the early release of toxin drugs and causing off-target toxicity, which affects the therapeutic window of PDC. After entering the target cell, the linker must ensure the effective release of the toxin drug to exert its killing effect.
Linkers can be divided into cleavable linkers and non-cleavable linkers. A cleavable linker can be cleaved enzymatically or chemically. Non-cleavable linkers cannot be activated by external stimuli. While cleavable linkers are better for developing targeted therapeutics, non-cleavable linkers are more stable in metabolic cycles in vivo. The choice of cleavable or non-cleavable linker depends on the design and mode of action of the targeted therapeutic drug.
Figure 4. The chemical structure of different linkers
(Image source: References )
Drug Toxins in PDC
Drug toxins are an integral part of the process of killing tumors. After PDCs enter the cells, drug toxins are the main cause of the final death of the target cells. Therefore, the toxicity and physicochemical properties of drug toxins can directly affect the ability of drugs to kill tumors, thus affecting its efficacy. Conjugated cytotoxins must have the following four requirements: clear mechanism of action, small molecular weight, high cytotoxicity, and retention of antitumor activity after chemical conjugation with peptides.
Each drug toxin has its limitations, such as poor pharmacokinetic properties. However, the drug's non-selectivity is the biggest disadvantage, causing serious side effects. These peptides allow specific targeting, thus broadening the therapeutic field. Due to the attachment of chemotherapy drugs to peptides, increased doses are often required to reduce the cytotoxic dose reduction. There are many criteria for determining the cytotoxicity of PDCs, such as in vivo circulation stability, high efficiency of drug effect, and the presence of linker-linkable sites. Chemotherapeutic agents in PDC include doxorubicin, paclitaxel, etc. In addition, it also includes radionuclides, such as 177Lu dotatate.
Stability of PDC
Similar to peptides, the major disadvantages of PDC are poor circulatory stability and rapid renal clearance. PDC should remain stable in circulation to prevent pre-release chemotherapy and systemic exposure. It has been confirmed that nanoparticles can enhance the stability of PDC. One approach to overcome the poor cycling stability is to combine PDCs with gold nanoparticles (AuNPs). The overall stability can be improved due to its desirable physicochemical properties, safety, relative ease of synthesis, and long circulating half-life. When PDC used to treat lymphoma cells in mice was combined with PEG-coated AuNP to generate selective PEG-AuNP-PDC, the PDC circulating half-life was increased 90-fold. Nanoparticles enhance the stability of PDCs through a bifunctional approach. The design principle is based on near-infrared light non-invasive anti-tumor therapy photothermal therapy (PTT), and nanomaterial-enhanced PK properties of PDCs is an ongoing research area, making PDCs have great potential for clinical trials.
Administration Route of PDC
PDC must be administered intravenously, similar to ADC. More research is needed on the delivery systems of peptides and proteins. Recently, a novel method for oral administration of peptides has been reported in the literature, using the peptide self-assembly method to prepare pectin dihydroartemisinin/hydroxymycin nanoparticles (PDC-H-NPs). Combining PDC-H-NPs with pro-pectin and anticancer drugs dihydroartemisinin or hydroxycamptothecin can increase drug loading, improve water solubility, and achieve drug release (Figure 5). However, the route of administration, particle size, pharmacokinetic properties, immune clearance, etc. hinder the clinical application of nanomedicine. There are also literature reports using lipid nanoparticles and cell-produced exosomes as drug delivery systems. In the future, oral administration may be a new strategy for using PDC drug candidates in clinical trials.
Figure 5. The properties of the peptide in vivo and in vitro
(Image source: References )
PDC is a combination of peptide and chemotherapeutic drug, combining the selectivity of peptide with the lethality of chemotherapeutic drug. By modifying the amino acid sequence of the polypeptide, PDC can change the hydrophobic and ionizing properties of the conjugate, which solves the problems of poor water solubility and fast metabolism. Compared with small molecules and biologics, peptides are rarely used clinically. However, they exhibit excellent versatility. PDC drugs can enhance the permeability of tumor cells, reduce immunogenicity and reduce development costs.
PDC, as an emerging research field in the fight against cancer, has its advantages compared with ADC, but there are still many difficulties to be overcome. Fortunately, based on ADC's experience, there may be some shortcuts and fewer detours in PDC research. At the same time, with the innovation of technology, PDC research will gradually be clinically verified, thereby promoting the development of this field and bringing more options for treatment.
Huateng Pharma is a leading and professional manufacturer which can provide PEG linkers for the R&D of PDCs. We are committed to promoting the progress of your PDC discovery and development projects. We offer the full range of PEG derivative development services and provide the most comprehensive media for conjugation research.
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