Accelerating Glycosylated Drug Discovery with Custom Glycopeptide Libraries

Why Glycopeptide Libraries Are Valuable in Early-Stage R&D?

(1) Mapping Glycan-Protein Interactions

Glycopeptide libraries can be used for screening in early stage R&D to map glycan-protein interactions. Glycans are involved in many biological processes including cell signaling, immune recognition, and binding of pathogens. Glycan-protein interactions will allow us to understand the molecular basis of biological processes and identify potential therapeutic targets. Glycan-binding proteins (GBP) include lectins and antibodies that bind to specific glycan motifs on cells or pathogens. The analysis of glycopeptide libraries helps determine which glycan motifs are bound by GBP. The acquired data allows scientists to create glycopeptides that modify GBP binding to target glycans by either increasing or decreasing this interaction. Glycopeptide libraries can be employed to measure binding kinetics and thermodynamics in glycan-protein interactions. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can be utilized to quantitatively measure glycan-protein binding affinities and specificities. Scientists can then modify glycopeptide structures for certain drug delivery and other therapeutic applications.

Fig. 1 Representation of immune networks mediated by glycans and glycan-binding proteins in autoimmunity and/or chronic inflammation.^1,2

(2) Discovering Functional Glyco-Epitopes

Glyco-epitopes are the glycan structures that can be recognized by the immune system. Glyco-epitopes can be targeted with vaccines and immunotherapies. Engineered glycopeptides can present tumor-associated glycan antigens (TAGs) that cancer cells frequently overproduce on their surfaces to develop cancer immunotherapeutics. Screening glycopeptide libraries with immune cells allows for identification of glyco-epitopes that elicit an immune response, which in turn allows for the design of glycopeptide-based vaccines for targeting cancer cells. Glycopeptides have already been shown to have potential as immunotherapeutic agents in preclinical studies. Glycopeptide libraries can be screened to identify the role of glyco-epitopes in autoimmune diseases. Autoantibody screening of glycopeptide libraries enables the identification of glyco-epitopes that these antibodies target along with an understanding of related disease mechanisms. The obtained data enables researchers to create glycopeptides that adjust immune responses through enhancement or inhibition of autoantibody-antigen binding.

Fig. 2 Glycans as a major connective chain that controls T cell response in either a tolerogenic or immunostimulatory scenario.^3,4

Key Considerations in Library Design

(1) Glycan Diversity (Tn, STn, Lewis, GlcNAc, Sialic Acid)

The library design incorporated various glycans to ensure its structural relevance to biological systems. The glycans incorporated in our glycopeptide library included Tn (GalNAc), STn (GalNAcα1-3Gal), Lewis antigens, GlcNAc and sialic acid. These glycans can be used for investigations of Lewis antigen interactions in inflammation and disease, aberrant glycosylation in cancer and glycan-directed cell signalling. Tn and STn antigens are targets of immunotherapy for cancer therapeutics, and are commonly overexpressed in cancer cells, whereas Lewis antigens play a role in inflammation and immune cell recognition. GlcNAc and sialic acid were also included in the library as they can also be present on the above glycans, and are also important in cell recognition and signalling.

(2) Peptide Scaffolds and Epitope Presentation

The selection of peptide scaffolds and epitope presentation is another factor to consider with glycopeptides. The presentation of glycans along with their accessibility to binding partners depends on peptide scaffolds. The glycan's conformation and orientation are influenced by the peptide scaffold which affect optimal binding with GBPs and antibodies. Flexible spacers and PEG-like linkers prove beneficial in boosting glycan exposure and accessibility. Peptide sequence itself can also affect the stability and immunogenicity of the glycopeptide. The structure of peptide scaffolds must be carefully designed so that glycans adopt the proper native conformation on cell surfaces while also being available to binding partners.

(3) Linker Length, Valency, and Solubility Optimization

Design of glycopeptide libraries is also guided by optimization of linker length, valency and solubility. The linker length is an important consideration since it will determine the position and accessibility of the glycan moiety. For example, short linkers may restrict movement of the glycan, increasing the possibility of steric clashes. Long linkers may allow for more flexibility and better presentation of the glycan. Valency of the glycopeptide is another critical design feature since avidity can greatly affect affinity of the glycopeptide. Multivalent glycopeptides have been used to take advantage of avidity effects in therapeutic and diagnostic applications. Solubility is another important factor, especially for biological assays and in vivo applications. Glycopeptides achieve better solubility and maintain their stability and function in aqueous environments when hydrophilic linkers or PEGylation are used as enhancement methods.

Our Custom Glycopeptide Library Services

(1) Focused Libraries for Receptor Binding Studies

The custom glycopeptide library service enables specialized libraries for receptor binding investigations. Focused libraries can be built around specific glycans such as Tn, STn, Lewis antigens, GlcNAc, and sialic acid, etc. that are known to bind to a variety of receptors and GBPs. Targeted glycopeptide libraries with glycan antigens like Tn, STn, Lewis, GlcNAc and sialic acid enable systematic studies of how these glycans interact with specific receptors including ASGPR or lectins within biological systems. Lectins are proteins that recognize and bind to specific glycan structures and are involved in numerous immune recognition and cell signaling events. For example, Tn and STn antigens are frequently overexpressed in cancer cells and are recognized by both antibodies and immune cells. Targeting these antigens with therapeutic antibodies is an area of active investigation in cancer immunotherapy. In addition to Tn and STn antigens, Lewis antigens are involved in inflammation and immune responses. The glycan structures GlcNAc and sialic acid are also involved in a variety of cell signaling and recognition events. These glycan structures can be mapped by using targeted glycopeptide libraries.

(2) Scaffold-Glycan Combinations with Defined Stereochemistry

Glycopeptide libraries for targeting glycan-protein interactions and glycan-specific recognition events. We provide glycopeptide libraries that encompass multiple scaffold-glycan combinations with specifically defined stereochemistry. The libraries provide fine control over glycan placement and orientation, allowing for targeted exploration of glycan-protein interactions and functional glyco-epitopes. Defined stereochemistry allows advanced synthesis techniques to attach glycans to specific peptide scaffold locations. N-linked glycans to asparagine and O-linked glycans bonded to serine or threonine with defined stereochemistry are potential glycopeptide structures researchers can obtain. The designed glycan attachment together with stereochemistry on glycopeptides allows for the enhancement or induction of targeted properties such as receptor affinity gains or immune responses.

(3) Delivered with QC, Documentation, and Optional Labeling

QC protocols have been established for our custom glycopeptide library services. Extensive analytical testing is carried out on each batch of glycopeptides. The identity and purity of the product by MS and HPLC are confirmed. This important validation ensures the glycopeptides will meet the specific requirements for receptor binding studies and other applications. We also provide extensive library documentation, including batch consistency reports and structural validation, to give researchers a better understanding of the glycopeptide composition in the library and the confidence to use the library in their studies. The custom fluorescent and biotin labeling services offered enable the customization of glycopeptides for specialized functional applications such as imaging and affinity purification.

Glycoamino acids we can provide

References

1. Image retrieved from Figure 1 " Representation of immune networks mediated by glycans and glycan-binding proteins in autoimmunity and/or chronic inflammation," Pinho S S.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Pinho S S.; et al. " Immune regulatory networks coordinated by glycans and glycan-binding proteins in autoimmunity and infection." Cellular & molecular immunology, 2023, 20(10): 1101-1113.
3. Image retrieved from Figure 1 " Glycans as a major connective chain that controls T cell response in either a tolerogenic or immunostimulatory scenario," Pereira M S.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
4. Pereira M S.; et al. " Glycans as key checkpoints of T cell activity and function." Frontiers in immunology, 2018, 9: 2754.
5. Chao Q, Ding Y, Chen Z H, et al. Recent progress in chemo-enzymatic methods for the synthesis of N-glycans[J]. Frontiers in chemistry, 2020, 8: 513. https://doi.org/10.3389/fchem.2020.00513.
6. Gao C, Wei M, McKitrick T R, et al. Glycan microarrays as chemical tools for identifying glycan recognition by immune proteins[J]. Frontiers in chemistry, 2019, 7: 833. https://doi.org/10.3389/fchem.2019.00833.