High Resolution 3D Epitope Mapping. Antibody-Antigen 3D Structure Services
SARomics Biostructures’ Contract Research Organization (CRO) team specializes in high-resolution 3D mapping of epitopes and paratopes, as well as the crystallization and structure determination of antibodies and antibody-antigen complexes. Our services are enhanced by our FastLane list of extracellular proteins, which facilitates antibody-antigen complex crystallization, and by our proximity to the MAX IV synchrotron in Lund.
High-Resolution 3D Epitope Mapping
SARomics Biostructures’ CRO team has extensive experience in high-resolution antibody and antibody-antigen structure determination, 3D epitope and paratope mapping, and higher-order structure analysis of biosimilars using X-ray crystallography and NMR spectroscopy. All our studies also include biophysical characterization of the proteins in solution using biophysical techniques. For details of our services, please see below:
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We have contributed to many projects by determining the three-dimensional structures of antibody-antigen complexes and epitope mapping. Several of the structures have been published in leading journals. Please see the list of case studies below. See our blog post on 3D epitope mapping techniques.
FastLane Proteins for Antibody-Antigen Complex Crystallization & 3D Epitope Mapping
X-ray crystallography and cryo-electron microscopy (cryo-EM) are the gold standards for high-resolution 3D epitope mapping. Currently, the Structural Antibody Database (SabDab) houses over 10,000 antibody structures and 9936 tertiary structures of antibody-antigen complexes determined by X-ray crystallography and cryo-EM. Of these, 6,570 structures were determined using X-ray crystallography, 3,945 using Cryo-Electron Microscopy, and 17 by NMR spectroscopy. Out of the X-ray crystallographic structures analyzed, 3,773 (57%) exhibited a resolution better than 3.0 Å, while 1,172 structures had a resolution exceeding 2.0 Å (18%). For cryo-electron microscopy (cryo-EM) structures, 1,475 had a resolution better than 3.0 Å (37%), and 397 structures achieved a resolution above 2.0 Å (10%). The statistics clearly demonstrate the superiority of these methods, which provide the most accurate high-resolution insights into antibody-antigen interactions, as well as detailed mapping of 3D epitopes and paratopes.
See our blog post on 3D epitope mapping techniques.
Several animal experiments have demonstrated that platelet glycoprotein VI (GPVI) plays a crucial role in thrombosis associated with ischemic stroke. On this basis, GPVI is a potential target for developing new antiplatelet molecules with low bleeding risk. The crystal structure of Glenzocimab, a Fab fragment of humanized anti-GPVI monoclonal antibody, with the monomeric extracellular domain of Platelet glycoprotein VI GPVI, has been determined at 1.9 Å. The structure revealed the Glenzocimab epitope at the dimerization site in the D2 domain of GPVI, the antibody binding to which inhibits GPIV interaction with CRP, collagen, and fibrin by disrupting dimerization, inducing conformational changes, and imposing steric hindrance.
Why Choose X-ray Crystallography for High-Resolution 3D Epitope Mapping?
A common misconception is that X-ray crystallography and cryo-EM techniques are too slow and expensive for 3D epitope mapping. However, advances in cryo-EM and X-ray crystallography instrumentation have greatly reduced costs and accelerated the determination of high-resolution structures of proteins and protein-ligand complexes. Our lists of FastLane proteins (see above), which include extracellular proteins ready for co-crystallization with their cognate antibodies, further reduce time and costs for high-resolution 3D epitope mapping. Today, it is fair to say that X-ray crystallography and cryo-EM are the most cost-effective and reliable techniques for determining the 3D structures of antibodies, antibody-antigen complexes, and 3D epitope mapping.
An important consideration is the type of epitope being studied. Epitopes are generally categorized into two main types: linear and conformational epitopes. Linear epitopes consist of short, continuous sequences of amino acids, whereas conformational epitopes are derived from the three-dimensional structure of the protein and do not have to form a continuous sequence within the antigen; these are three-dimensional epitopes. For high-resolution mapping of 3D epitopes, X-ray crystallography and cryo-EM are considered the gold standard due to their ability to provide high-resolution insights into intermolecular interactions and their high level of accuracy and reliability (see the statistics above for the SabDab database). In addition, they can reveal possible conformational changes and allosteric effects that occur during the formation of an antibody-antigen complex. Needless to say, this may significantly enhance antibody targeting.
Our blog post compares the techniques used for 3D epitope mapping with the benefits of experimental structural biology methods such as X-ray crystallography and cryo-EM. See also the case studies described below, which include some antibody-antigen complex structures determined at high resolution by the SARomics team using X-ray crystallography.
Case Studies: Antibody-Antigen Complex Structures Contributed by SARomics Biostructures
SARomics Biostructures CRO team has contributed to the 3D structure determination of numerous antibody-antigen complexes. Several of these projects have been published in high-ranking journals, including PNAS, Nature Communications, Cell Reports, iScience, Cancer Therapy, Blood Advances, and Structure.
Some of the papers are listed below; more details on the publications can be viewed in the PDF on the right or downloaded here.
Structural analysis of light chain-driven bispecific antibodies targeting CD47 and PD-L1. Malinge (Light Chain Bioscience – Novimmune SA) et al., 2024, mAbs, 16, 2362432.
In this study, the authors developed a bispecific antibody format that, unlike natural antibodies, which primarily use the heavy chain to interact with their specific antigens, utilizes the light chain (LC) for antigen recognition and binding. To enhance the understanding of epitope-paratope interactions, SARomics Biostructures assisted in the crystallization and structure determination of complexes formed by two antigen-binding Fab fragments with human CD47 and PD-L1 (see our FastLane™ Premium library). The analysis of these complex structures revealed that, while the light chains (LCs) play the dominant role in antigen binding, the heavy chain (HC) also engages in some interactions with both CD47 and PD-L1.
PDB ID: 8RP8 and 8RPB at a resolution of 2 Å and 2.79 Å, respectively.
Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies. Ferrari (Autolus Therapeutics) et al., 2024, Nat Commun, 15.
Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor β-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target. Here, the authors demonstrate specificity redirection by rational design, using structure-guided computational biology to generate a TRBC2-specific antibody (KFN), complementing the previously described laboratory antibody with unique TRBC1 specificity (Jovi-1) for targeting a broader spectrum of T cell malignancies that clonally express either of the two chains. SARomics Biostructures assisted the project by determining the 3D structure of Jovi-1 in complex with TRBC2. The structure of the antibody-antigen complex enabled the explanation of Jovi-1’s high selectivity for TRBC1.
PDB ID: 7AMP, 7AMQ, 7AMR, and 7AMS at a resolution of 2.64 Å, 2.35 Å, 1.95 Å, and 2.42 Å, respectively.
