Epitope Mapping for Drug Discovery

Analysis of antibody-antigen complexes plays a pivotal role in advancing our understanding of the human immune system. Insights into these complexes enable scientists to develop innovative therapies and diagnostics to combat disease. Epitope mapping is the process of identifying and characterizing the specific regions on an antigen to which antibodies bind. This information is crucial for understanding immune responses (specificity, cross-reactivity and immunogenicity), designing vaccines, developing targeted therapies (rational antibodies design), developing diagnostic tests and patent filing and protection.

Most interactions between antibodies and antigens rely on discontinuous epitopes, or conformational epitopes: these are formed by amino acid residues that are spatially close to each other in the folded protein but may not be adjacent in the primary sequence, and are only recognized by antibodies when the antigen is folded into its correct three-dimensional structure. There are several methods used for conformational epitope mapping, including Hydrogen–Deuterium Exchange (HDX), deep mutational scanning, X-ray crystallography (XRC), and Cryogenic Electron Microscopy (Cryo-EM). While XRC has traditionally been the gold standard for understanding epitopes, HDX and deep mutational scanning can provide fast results, and cryo-EM is quickly gaining traction for its ability to map specific atomic interactions at approximately 3.5 Å, as well as its ability to visualize large, flexible, and dynamic molecules that do not easily crystallize.

Rapid Results with Hydrogen–Deuterium Exchange & Mass Spectrometry (HDX-MS) and Deep Mutational Scanning 

Hydrogen-Deuterium Exchange (HDX) coupled with Mass Spectrometry (MS), is an effective method to rapidly supply information for epitope mapping. This method analyzes the solvent accessibility of various regions of the antigen and antibody, correlating reduced solvent accessibility with the regions forming the protein-protein interfaces. If the three-dimensional structure of the antigen is known, mapping of these protected regions onto the 3D representation can rapidly provide valuable insights into the general area of interactions. It can also reveal the dynamic conformational changes that occur during the complex formation process.

Deep mutational scanning is a high-throughput experimental technique used to systematically study the effects of mutations on a protein or other biomolecules. It allows researchers to analyze the functional consequences of a large number of mutations across the entire sequence of a gene or protein in a single experiment. The power of this method is scale, as tens of thousands of mutants can be assessed in a mixed pool.

It is important to note that both methods described above primarily offer "low-resolution" details (10-50 Å) about the general area of the antigen-antibody interaction, even if  this limitation is being improved by new technology advancements. While they can provide valuable additional information about the overall conformational changes occurring upon antibody-antigen binding they do not really provide detailed information about the precise atomic interactions on a residue-to-residue level that may be necessary to enable rational antibody design or strengthen patent claims.

Moving into atomic resolution with X-ray Crystallography (XRC)

Three-dimensional epitope mapping, the determination of  the three-dimensional structures of the antibody-antigen complex, provides detailed insights into the spatial arrangement and specific amino acid interactions involved in the binding interface. Three-dimensional epitope mapping can help in precise epitope identification, can define the atomic details of the antibody specificity, can guide the rational design of new antibodies with specific properties and can strengthen patent claims by providing detailed information about the unique aspects of an antibody binding to its target antigen (novelty, specificity, and utility of the invention), increasing the chances of successful patent applications.

XRC is considered the traditional method that offers atomic-resolution insights into antibody-antigen interactions with a relatively small amount of material. XRC allows scientists to determine the three-dimensional structure of the antibody-antigen complex by analyzing the diffraction patterns generated by crystals of the complex of interest.  While XRC can provide high resolution information in a short amount of time, it has limitations when it comes to samples that do not crystallize or are large, dynamic, or flexible. In the specific case of epitope mapping, intact antibodies are not easily amenable to crystallization (they are large and flexible), as is the case for FABs generated by digestion of the parent MAB (heterogeneous composition).  The size and flexibility of the antigen can also play a role in the crystallizability of the sample and the possibility of getting high resolution data.

Partnering with NanoImaging Services to Map Antibody Epitopes through Cryo-EM

While traditional methods such as X-Ray Crystallography and Hydrogen-Deuterium Exchange have some advantages, Cryo-Electron Microscopy (Cryo-EM) has emerged as a breakthrough technique, with the capability of providing density maps at a resolution of ~3.5 Å or better, enough to allow for the identifications of residue-based details. Cryo-EM can be used for a variety of biological molecules,  even for large complexes, including those of antigens bound to full antibodies providing an exceptional advantage over other techniques.

Since cryo-EM is not hindered by the crystallization challenges faced by XRC or the limitations of sample size and dynamics encountered with HDX,  it can potentially enable the precise mapping of epitopes in even the most challenging cases.

Researchers have a wide range of tools available to them, and each technique can yield valuable insights into antibody-antigen interactions. Identifying the optimal technique for your specific sample requires a comprehensive understanding of the limitations of each technique and their varying suitability for different sample types.

At NIS, we understand the significance of this decision, and are here to collaborate and partner with you. Our team of experts is dedicated to providing the highest level of expertise and assistance in selecting the techniques that will best serve your project goals. Together, we can explore the complexities of structural biology and reveal the valuable insights that lie within antibody-antigen interactions.