Solving the Top 3 Challenges in Glycopeptide-Based Drug Delivery Systems

Challenge 1-Poor Conjugation Efficiency

(1) Choosing the Right Linker Chemistry for Glycopeptides

Conjugation linker chemistry remains a critical component in drug delivery systems. Because various linkers directly impact conjugation stability and efficiency, the choice of linker is an important one to make early on. The selection of linkers ensures stable glycopeptide (GP) conjugation to therapeutic delivery vehicles yet permits payload release at the action site. Non-cleavable linkers are typically more stable and the payload remains conjugated until after cellular uptake and intracellular degradation. Cleavable linkers are typically designed to be cleaved in response to some stimuli, like changes in pH, redox potential, or enzymatic activity. For example, pH-sensitive linkers can be cleaved in the low pH of the tumor microenvironment (pH 4.5–6.5). On the other hand, enzyme-sensitive linkers can be cleaved by enzymes that are overexpressed in cancer cells. Linkers are also optimized for minimal steric hindrance and appropriate orientation of the GP to enable optimal conjugation. The overall hydrophilicity of the conjugate should also be considered when selecting a linker, as this can affect pharmacokinetics and cellular uptake.

(2) Avoiding Steric Hindrance Around Glycosylation Sites

Glycopeptides function requires unobstructed glycosylation sites because steric hindrance from bulky substituents or the conjugated payload blocks target receptor binding. The design of linkers must account for glycosylation site orientation and the attached payload to reduce steric hindrance effects. A method involves flexible linkers which create the necessary separation between the GP and the therapeutic agent. Polyethylene glycol (PEG) spacers or other hydrophilic linkers can be used to introduce space and flexibility between the two. PEGylation of classical dipeptide linkers have been found to increase the therapeutic index and decrease non-specific binding. Another strategy is to optimize the orientation of the glycosylation sites relative to the linker.

Challenge 2-Rapid Clearance or Enzymatic Degradation

(1) Optimizing Glycan Structure for Stability

Glycopeptides face major obstacles in drug delivery because they undergo rapid clearance and enzymatic degradation. Rapid clearance and enzymatic degradation are two ways in which the efficacy of the therapeutic can be reduced, either by not allowing the GP to reach the target or by causing it to degrade before it can be effective. The glycan can be engineered to improve the stability of the GP and minimize these two possibilities. Glycans can have various effects on the stability of GPs. N-linked glycans exhibit greater stability than O-linked glycans, the former can be more resistant to enzymatic degradation than the latter. This is because N-linked glycans are attached to the amide nitrogen of asparagine residues, whereas O-linked glycans are attached to the hydroxyl groups of serine or threonine residues. Asparagine-linked glycans can also be more protected from enzymatic attack because of the presence of the amide nitrogen, which can create additional steric hindrance. The complexity and branching of glycans can also impact their effect on stability. Highly branched and complex glycans have been shown to improve the stability of GPs, as they can provide multiple points of interaction with the peptide backbone and reduce the likelihood of enzymatic cleavage. GPs with complex N-linked glycans, for example, have been shown to have longer circulation times and lower clearance rates than those with simpler glycans.

Fig. 1 Protein glycosylation.^1,2

(2) Incorporating PEG-like Modifications for Half-life Extension

It is thought that the large PEG merely sterically occludes the protein from proteases and antibodies. In many cases, a therapeutic PEG-protein conjugate will be less active than its non-PEGylated counterpart in in vitro biochemical assays, presumably because the attached PEG also sterically hinders access of substrates or binding partners to the protein surface/active site. Extended serum half-life of PEG-protein conjugates often compensates for reduced activity levels thus enabling less frequent dosing while reducing side effects when compared to non-PEGylated proteins. The attachment of PEG enhances proteolytic stability, helps to ameliorate immunogenicity, increases resistance to bacterial-secreted enzymes, prolongs blood circulation half-lives and can improve biodistribution and drug bioavailability. Based on N-terminal PEGylation and glycosylation, two conjugated derivatives of the antibacterial HDP IDR1018 were designed to improve biocompatibility and biological characteristics of the original peptide sequence. Further studies are needed to optimize this approach by using different glycosylated parts, different PEG chain lengths and different ligation methods. However, the current conjugation resulted in a significantly reduced enzymatic degradation, hemolysis and cytotoxicity, and in an increased half-life. Importantly, pegylation did not impede the immunomodulatory properties of the peptides, while glycosylation generated an excellent immunomodulatory conjugate that can strongly stimulate the release of chemokine MCP-1 and anti-inflammatory cytokine IL-1RA, and simultaneously inhibit the production of pro-inflammatory cytokines TNFα and IL-1β induced by LPS.

Challenge 3 – Lack of Target Specificity

(1) Glycan-Receptor Mapping Tools

The primary concern of GP delivery strategies is off-target effects due to the absence of target specificity. Glycan-receptor mapping tools (GRMTs) can be used to design and synthesize GPs with desired properties for drug delivery purposes. For example, by using GRMTs, researchers can identify specific glycan motifs that are recognized by target receptors and design GPs that incorporate these motifs to enhance target specificity. Lectin and antibody arrays can be used for high-throughput screening of glycan-receptor interactions to help define carbohydrate specificity and identify ligands. MS can be used to characterize the binding of glycans to receptors and to identify the specific glycan structures recognized by these receptors, which can then be employed to design GPs with improved target specificity.

(2) Designing Multi-Valent Glycopeptides for Better Binding

A multi-valent GP design approach for improving target specificity of GP-based delivery can be highly effective. In the context of this review, multi-valency is the presentation of two or more glycan ligands on a single scaffold. The objective of the multi-valent design approach is to improve the binding affinity and/or specificity via avidity effects. Most GBPs display an affinity for clustered or multimeric glycans, and as such the design of GPs with multimeric scaffolds may improve GP targeting. GPs can be designed with multimeric scaffolds to improve their binding affinity to target receptors. For example, O-glycocarriers can be designed to display a cluster of Tn or STn glycans on a multimeric scaffold to improve the binding of the GP to a target receptor, such as the asialoglycoprotein receptor (ASGPR). In this case, this multimeric approach could improve target specificity as well as reduce off-target effects as glycans presented on linear or non-multimeric scaffolds would not be able to bind to ASGPR. Multi-valent GPs can be used as functional probes to identify and track glycan-binding receptors. For instance, GFP-tagged O-glycocarriers with displayed Tn glycans (O-glycopeptides) have been used as probes for uptake studies into human liver HepG2 cells. Research into GP interactions utilizes these probes to investigate target receptor engagement and to understand GP uptake and trafficking mechanisms.

Our Solution: Tailored Glycopeptide Design and Synthesis

(1) Support from Scaffold Design to Final Purification

We create glycopeptides that meet your needs through our flexible synthesis technology. Our team possesses extensive experience in scaffold engineering to develop glycopeptides with the required structure and functionality. Our computational software and modeling techniques enable us to produce scaffolds that enhance glycan and peptide display through the careful selection of glycans and their placement on peptides with optimal structural design. We can engineer scaffolds that increase the glycopeptide's binding strength to particular receptors like ASGPR which cancer cells tend to overexpress, and tailor our synthesis method according to your needs and create a broad range of glycopeptide constructs by integrating various glycans and peptides. Our lab specializes in chemical and enzymatic glycosylation procedures which result in precise glycosylation products with high yield and purity. We can also provide conjugation services to link glycopeptides to other moieties, such as drug molecules or fluorophores, to yield multifunctional constructs.

(2) Batch Consistency and Delivery Format Flexibility

Quality is our highest priority and we have strict quality control (QC) procedures in place to ensure the quality and reliability of our products. To maintain batch-to-batch consistency we applied strict quality control measures throughout each stage of synthesis to purification. We have detailed records of synthesis and purification conditions that allow for high fidelity batch reproduction. Our strict QC procedures ensure that our glycopeptides meet the highest standards of purity, composition, and functionality. We deliver glycopeptides available as solutions, lyophilized powders and conjugated forms. This ensures that the glycopeptides are available in the desired form for immediate use in your experiments or formulations.

Peptide Conjugation Services at Creative Peptides

References

1. Image retrieved from Figure 1 "Protein glycosylation," Ma B.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Ma B.; et al. "Protein glycoengineering: an approach for improving protein properties." Frontiers in Chemistry, 2020, 8: 622.
3. Mastrangeli R, Satwekar A, Bierau H. Innovative Metrics for Reporting and Comparing the Glycan Structural Profile in Biotherapeutics[J]. Molecules, 2023, 28(8): 3304. https://doi.org/10.3390/molecules28083304.
4. Nguyen D N, Xu B, Stanfield R L, et al. Oligomannose glycopeptide conjugates elicit antibodies targeting the glycan core rather than its extremities[J]. ACS central science, 2019, 5(2): 237-249. https://doi.org/10.1021/acscentsci.8b00588.