High Resolution Epitope Mapping with Cryo-EM

Leveraging the power of cryo-EM for epitope mapping

Epitope mapping plays an important and increasingly critical role in many facets of antibody and vaccine development. Referring to the process of identifying the interface between the paratope of an antibody and the site on the antigen (epitope) to which it binds, epitope mapping can return valuable information.

At high resolution (for the sake of argument at resolutions where amino acid side chains can be unambiguously assigned), epitope mapping can:

• Directly inform optimization strategies for rational in silico design
• Distinguish between antibodies with highly similar epitopes
• Identify conformationally-specific antibodies
• Support patent filings
• Identify surface features, like post translational modifications, that may require alteration if located near the epitope

More traditional epitope mapping approaches include negative stain TEM, HDX-MS, mutagenesis scanning and X-ray crystallography. However, limitations in resolution, sample requirements, time to "map" and conformational dynamics in these methods have limited their utility in broader antibody and vaccine campaigns.

Advancements in the cryo-EM single particle analysis workflow have uniquely positioned the method as an emerging gold-standard for rapid epitope mapping studies at moderate to high resolution.

Benefits of Cryo-EM:

Challenging antigens: Cryo-EM is uniquely positioned to study large, flexible, and/or dynamic systems, along with integral membrane proteins. The predominant requirement is that the ordered mass of the antigen at the epitope-paratope interface be of sufficient size. Ideally, the antigen will contribute more than 50 kDa of ordered mass which combines with that of the ~52 kDa Fab to produce a "local" mass of 100 kDa or more. Like a snow angel, the extremities of the antigen and the antibody can wave about and with details washing away during averaging, leaving a well-ordered body or mass around the epitope.

Skipping Fab generation: The production of Fabs can be challenging and time consuming, especially when material is limiting, resources have been outsourced, or the overall timelines of the program are aggressive. Cryo-EM workflows can use mAbs directly without requiring the production of purified Fab fragments.

Throughput: When multiple, non-competing antibodies are available for an antigen, multiplex experiments are possible. A single complex of the antigen bound to multiple mAbs/Fabs is assembled. Standard vitrification and data acquisition workflows are then undertaken. Individual epitopes are the mapped during data processing and 3D reconstruction. This approach can also be used when the ordered mass of the antigen is small and would benefit from the addition of a high affinity binder.

Sample considerations:

Ordered Mass of the Antigen: Larger, conformationally homogenous systems are preferable, in line with other typical recommendations for straightforward cryo-EM efforts. Improvements in sample preparation, data acquisition and data processing, however, are continuously lowering mass limits for the method. In certain circumstances, antigens much smaller than 100 kDa can be efficiently mapped.

Antibody-Antigen Affinity: Cryo-EM allows us to work with much weaker affinity pairs than traditional negative stain protocols, as the protein concentrations typically employed during sample vitrification are much higher. That said, high affinity, homogenous systems greatly reduce the complexity of the workflow.

Antibody behavior: It is easy to forget that antibodies are themselves proteins and thus are not all equally well behaved. They vary in degree of flexibility, propensity for aggregation, etc. Fab fragments can be preferable, as they reduce complexity of the system and minimize other spurious behaviors that can impede structure determination.

How fast is cryo-EM enabled epitope mapping?

For a well-behaved Ab-Ag pair, epitopes in the 3-3.5 Å, range are possible within 24-48 hours of the start of an experiment. In a typical workflow, sample preparation conditions are screened throughout the day. This involves optimizing sample concentration, grid supports, additives and vitrification parameters. Live data processing is used in these short sessions to provide in-depth assessment of the “promise” of each condition. Data are acquired overnight on either the Thermo Fisher Scientific Glacios-Falcon 4 or Thermo Fisher Scientific Krios-K3 instrument configuration. CryoSPARC Live ensures the data is ready for careful inspection on completion with initial maps ready soon afterward.

If the most useful/desirable resolution is not achieved in this first pass, the comprehensive first-look and initial maps offer tremendous insights for immediate next steps to optimize the protein system and/or cryo-EM parameters for further improvements.

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