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Leveraging cryo-EM for antibody discovery and epitope mapping
Large-molecule antibody biologics have revolutionized drug discovery owing to their superior target specificity and amenability to versatile engineering. As of 2023, more than 160 monoclonal antibody therapies have been approved by at least one regulatory agency in the world for a wide range of indications. The approved antibody therapies include canonical antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, antibody fragments, immunoconjugates and Fc-Fusion proteins. Cryo electron microscopy (cryo-EM) has emerged as a crucial tool in accelerating the entire process, starting from protein target selection to the development of therapeutic antibodies.
Epitope mapping with cryo-EM
The goal of an antibody discovery pipeline is to develop a stable antibody drug with required specificity, selectivity and affinity that can be patented and/or progressed to clinic. Epitope mapping is an established process used in multiple stages of the antibody discovery workflow that provides information about the antigen-antibody interaction and helps to understand the mode of action. Detailed epitope information is important to strengthen intellectual property (IP) claims.
Epitopes can be classified into two groups - Linear epitopes, and conformational epitopes. Although, there are several technologies available to characterize the epitope-paratope interaction, cryo-EM offers several advantages over these technologies:
- Accurate epitope mapping of the epitope and the paratope at a single amino acid level, even of challenging and/or flexible targets.
- Determine the spatial arrangement of discrete and overlapping epitopes (both linear and conformational) on the large antigen surface.
- The technique does not require labeling or immobilization and is free from crystallization artifacts.
- Provides structural insights early on, driving faster decision-making which saves both cost and time.
More than one epitope can be found on an antigen.
Cryo-EM is a rapid, robust, versatile, and widely available method that supports the antibody discovery and development process. The role of cryo-EM based structure biology can be grouped into three categories based on the antibody function.
Epitope mapping to develop therapeutic antibodies
Epitope mapping using cryo-EM offers high-resolution information at the level of a single amino acid. Such detailed information can help elucidate the mode of action of antibody therapeutics. Having a structural basis of the antigen-antibody interaction can lead to the design of modular antibody formats like bispecific antibodies and ADCs.
Immunogenicity profile and epitope mapping to develop effective antibodies
Cryo-EM Polyclonal Epitope Mapping (Cryo-EMPEM) is growing in popularity to study the immune response against biologics such as antibodies and vaccines. This workflow allows you to study the polyclonal antibody response against an immunogen/vaccine candidate. Antigen bound to these antibodies can directly be isolated from the serum and subjected to high-resolution cryo-EM analysis. Detailed structural information allows the mapping of different epitopes (antigenic part) on the immunogen (vaccine/therapeutic antibody). These epitopes can then be engineered to enhance the immunogenicity for vaccine design and reduce the immunogenicity for antibody therapeutics and gene therapy viral vectors.
High resolution CryoEMPEM mapping at the amino acid level of neutralizing antibodies binding to different epitopes. Image adapted from Antanasijevic A et al 2021.
Suggested further reading:
- Structural mapping of antibody landscapes to human betacoronavirus spike proteins, Science Advances (2022).
- Mapping Polyclonal Antibody Responses in Non-human Primates Vaccinated with HIV Env Trimer Subunit Vaccines, Cell Reports (2020).
- Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM, Nature Communications (2021).
Epitope mapping to develop antibodies as use as a tool or reagent
Due to their remarkable selectivity, antibodies are routinely used to develop highly specific diagnostic tests, and affinity resins are used in bioprocessing and downstream purification. More recently, antibody-derived fragments are also being used as a tool to study other therapeutically relevant target proteins such as GPCR and Ion channels. Some of these tools are nanobody, megabody, legobody and NabFab.
Cryo-EM density of solute transporter PepT2-nanobody complex. Image adapted from Parker J et al 2021.
Cryo-EM structure of a complex of RBD domain of the SARS-CoV-2 spike protein and Legobody. Image adapted from We X and Rapoport T, 2021.
Antibody discovery workflow
The typical antibody discovery workflow is a long and tedious process that involves multiple steps to develop a successful antibody candidate. Of all the antibody therapeutics that enter phase 1 clinical trials, only 22% go on to receive regulatory approval. Structural information afforded by cryo-EM early in the discovery and development process is an important accelerator of this process. This information can drive structural modification to improve the developability and alter antibody properties in informed manner. Later in the pipeline, cryo-EM can be used to structurally characterize immunogenicity of candidate antibody in preclinical models and clinical trial stages. Below, we highlight where structural information can drive faster and better-informed decision making.
- Hit to lead
- Lead optimization
- Candidate selection
- Clinical trials
Hit to lead in therapeutic antibody development
When selecting lead antibody candidates from a large hit dataset, structural information provides detailed epitope binding information and informs on the developability aspect of the antibody molecule. Cryo-EM can help identify the most promising lead antibodies for lead optimization.
Structure elucidation by cryo-EM provides:
- Resolution of the spatial arrangement of discrete and overlapping epitopes (both linear and conformational) on the large antigen surface
- Accurate mapping of both the epitope and the paratope at single amino acid level thus providing information on mode of action.
- Quick turnaround of epitopes at moderate to high-resolution with well resolved antigen-antibody interfaces.
When AI/ML approaches are applied to generate lead antibody candidates, cryo-EM generated structural data will not only help validate those candidates but also provide rich data to train the existing algorithms and drive downstream optimization of identified lead candidates.
Lead optimization in antibody development
During this critical step in the workflow cryo-EM data allows structure-guided engineering to optimize the properties of the antibody molecule. Structure-guided modification of lead antibodies ensures that modifications are introduced at advantageous positions and that such modifications do not cause unwanted structural or behavioral changes of lead candidates.
Molecular dynamics simulations are routinely used to model antigen-antibody interfaces. Success of such analysis is influenced by the quality and accuracy of the starting structural models. Rapid elucidation of cryo-EM structure of the target antigen-bound antibodies provides optimal starting point for subsequent in silico modeling and engineering.
Candidate selection for therapeutic antibody development
Once the lead antibody candidates are identified, detailed epitope mapping can be performed. This will provide high resolution information on the mode of action (MoA) and ensures the most suitable candidate is then selected for clinical development. Moreover, rich data packages that include structural information of the target and candidate antibody help optimize antibody discovery pipelines and add value to the intellectual property (IP) claims. This level of detail fulfills the requirements for identification of specific amino acids responsible for antibody-antigen binding establishing novelty by clearly demonstrating that the antibody is targeting a unique epitope, and non-obviousness for antibodies that bind to a conformational epitope of discontinuous residues.
Clinical trials
Even after careful design of a new therapeutic antibody candidate the safety and immune response profiles much be understood. For example, antibody therapeutics can elicit an undesired immune response that reduce the bioavailability of the therapeutic and causes inflammatory or toxic responses. The use of cryo-EM to structurally characterize the immune response against the therapeutic antibody allows for optimization of the asset and/or development of next-generation variants. Furthermore, a comprehensive data trail of structural characterization through R&D and lead optimization adds rigor preferred by regulatory authorities.
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Resources
Cryo-electron microscopy is revolutionizing rational drug discovery pipelines
Rational design leveraging routine, high resolution protein structure determination is driving the discovery and development of diverse biologic and small molecule therapies. Cryo-EM delivers rapid epitope mapping on the atomic scale for antibody therapeutics and immune response profiling, supports elucidation of mechanism of action and is also enabling more therapeutic targets than ever before for Structure-Based Drug Design. Whether to modulate binding affinities or to optimize drug stability, all of these questions can be answered in just one day of data collection.
Watch the webinar to hear experts from Sanofi to discuss:
- An overview of how to quickly go from protein to structure
- Key examples of multi-specific drugs and structural insights of a CEACAM5-targeting antibody drug conjugate (ADC)
- How the adoption of Cryo-EM has significantly increased the number of the targets that can be identified for clinical trials in both biopharma and biotech
For Research Use Only. Not for use in diagnostic procedures.