SIAT^® In Vitro Class II HLA Binding Assay Services

SIAT® In Vitro Class II HLA Binding Assay Published Data FAQ Resources

Although various in silico models have been developed to predict the binding affinity of peptides in biotherapeutic drug candidates to class II human leukocyte antigen (HLA, also called major histocompatibility complex, MHC) alleles, the results of these prediction algorithms is not always in accordance with physical binding data. Therefore, after having defined the epitopes of interest in silico, further assays are needed to validate these predictions, among which class II HLA/MHC binding assay is most widely used.

SIAT® In Vitro Class II HLA Binding Assay

Creative Biolabs offers SIAT® in vitro class II HLA/MHC binding assay service that directly measures the affinity of predicted epitope peptide sequences to HLA alleles. The false positive and negative results generated by in silico models can therefore be eliminated. The information of immunogenic peptides identified by this assay is of great value in the development of novel biotherapeutic drugs. Based on the results of this assay, protein engineering strategies to deplete these T cell epitopes can be used to avoid side effects and the generation of anti-drug antibodies (ADAs).

Typically, an overlapping peptide library from a known protein of interest is generated by chemical synthesis. Afterward, a number of different HLA/MHC binding assay methods, including competition binding assay, direct binding assay, and real-time kinetic binding assay are used to determine the binding affinity of each peptide to one or more class II HLA/MHC alleles.

Competition binding assay

Peptides of interest are tested for their ability to compete against labeled high-affinity control peptides for binding to HLA/MHC molecules. IC₅₀ data is calculated by analyzing the dose-response curve. The binding assays can be performed for a broad representation of class II HLA/MHC molecules.

Direct binding assay

This assay is based on measuring of the ability of each peptide to stabilize the HLA/MHC-peptide complex, which will keep the native conformation if the binding affinity of tested peptide is high enough. A known T cell epitope is used as a positive control, and each peptide will be given a score by testing versus the positive control peptide. Currently, up to 60 DR, DQ and DP alleles and several mouse alleles are available for this assay.

Real-time kinetic binding assay

This assay can give kinetic information about the on- and off-rate at which each peptide interacts with HLA/MHC molecules in real-time. It can provide a complete information of whether a peptide could be presented long enough to be a good T cell epitope. For example, peptides with fast on- and off-rate may not be suitable candidate T cell epitopes. Protocols based on fluorescence polarization or surface plasmon resonance (SPR) are employed.

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Published Data

Validation In Silico Predictions by Molecular Modelling and Docking Analysis

Fig. 1 Pictorial representation of docked structure of P2 peptidewith HLA-DRB1*01:01 A in surface. (Nazam Khan, 2022)

The study investigates the prediction of T-cell and B-cell epitopes from Plasmodium vivax vaccine candidate antigens using in silico methods, aiming to identify promiscuous peptides that bind with diverse HLA Class-II alleles. This approach is significant because it addresses the challenge of genetic variability in both the parasite antigens and human immune response genes, which has hindered effective vaccine development against malaria. The research results demonstrated the successful identification of specific peptides with high binding affinity to multiple HLA Class-II alleles, suggesting their potential as broad-spectrum vaccine candidates. In the application of in vitro class II HLA/MHC binding assays, the study emphasized the use of molecular docking to validate the in silico predictions. This method confirmed the immunogenic potential of selected peptides by demonstrating their strong binding affinities, thus providing a robust foundation for further experimental validation and development of epitope-based vaccines. This integration of computational predictions with experimental validation techniques exemplifies a comprehensive approach to vaccine design, particularly for pathogens with high antigenic diversity like Plasmodium vivax.

Reference

Khan, Nazam, et al. "In silico study to predict promiscuous T cell and B cell-epitopes derived from the vaccine candidate antigens of Plasmodium vivax binding to MHC class-II alleles." Journal of Vector Borne Diseases 59.2 (2022): 154-162.

FAQ

What is the purpose of in vitro class II HLA/MHC binding assays in biotherapeutic drug development?
Class II HLA/MHC binding assays are used to predict the immunogenicity of biotherapeutic drugs. By assessing how drug peptides bind to HLA (Human Leukocyte Antigen) molecules, researchers can determine the likelihood of a peptide eliciting an immune response. This is crucial for ensuring the safety and efficacy of biotherapeutics, as unwanted immune reactions can lead to adverse effects.
How do class II HLA/MHC binding assays differ from class I assays in their application to drug development?
Class I HLA/MHC assays primarily focus on the binding interactions that occur within cytotoxic T lymphocytes, which are important for antiviral and antitumor responses. In contrast, class II HLA/MHC binding assays involve helper T cells, which play a significant role in orchestrating the immune system's response to larger pathogens and are crucial in the context of antibody production. This makes class II assays particularly important for evaluating drugs intended to modulate immune system functions or treat autoimmune diseases.
What are the common methodologies used in class II HLA/MHC binding assays?
Several methodologies are employed, including competitive binding assays, where peptides compete with a labeled reference peptide for binding to an HLA molecule, and direct binding assays, where the peptide of interest is directly labeled and its binding to HLA molecules is measured. Fluorescence polarization and surface plasmon resonance are also frequently used to quantify the binding affinities and kinetics.
Can class II HLA/MHC binding assays predict the clinical immunogenicity of biotherapeutics?
While these assays provide valuable insights into the potential immunogenicity of peptides, they do not fully predict clinical outcomes due to the complexity of human immune responses. However, they are an essential part of a comprehensive immunogenicity assessment strategy, helping to identify potentially immunogenic peptides early in the drug development process. Further validation in clinical trials is necessary to fully understand the immunogenic potential of a biotherapeutic.
What types of molecules can be tested using class II HLA/MHC binding assays?
Class II HLA/MHC binding assays can test a variety of molecules including peptides, small proteins, and fragments of larger therapeutic proteins. These assays are particularly relevant for molecules that are intended to interact with the immune system or that could potentially be immunogenic. This includes monoclonal antibodies, fusion proteins, and other biologics used in treating diseases such as cancer, autoimmune disorders, and inflammatory diseases.
How does the allelic diversity of HLA molecules impact the results of class II HLA/MHC binding assays?
HLA molecules are highly polymorphic, meaning there is a significant variation in HLA alleles among different individuals. This diversity can greatly influence the binding affinity of peptides to HLA molecules, impacting the predictive accuracy of these assays. To address this, assays typically include a panel of common HLA alleles to provide a broader understanding of a peptide's potential immunogenicity across diverse populations.
How do results from class II HLA/MHC binding assays influence further drug development decisions?
The results from these assays help identify peptide sequences within the biotherapeutic that might bind strongly to class II HLA molecules, suggesting a higher risk of triggering an immune response. Such findings can lead to the modification of the peptide sequence to reduce immunogenicity, or they may influence the decision to proceed with certain peptides in clinical trials. Additionally, these results can guide the design of further immunogenicity tests, including T-cell proliferation assays and cytokine release assays, to comprehensively evaluate the immune response.