High Resolution Epitope Mapping Services, Higher Order Structure Analysis
SARomics Biostructures’ CRO team has proven expertise in high-resolution epitope and paratope mapping services, analysis of antibody and antibody-antigen complex crystal structures, and NMR higher-order structure (HOS) comparability analysis for biosimilars.
We have contributed to many projects by determining the three-dimensional structures of antibody-antigen complexes, several of which have been published in leading journals. Please see the list of case studies below.
High-Resolution Epitope Mapping & Antibody-Antigen Structure Services
SARomics Biostructures’ CRO team has extensive experience in high-resolution antibody and antibody-antigen structure determination, 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:
- Crystallization and 3D structure determination of antibodies and antibody-antigen complexes by X-ray crystallography.
- High-resolution epitope and paratope mapping and analysis of antibody mode of action, utilizing X-ray crystallography (see the list of case studies below).
- Structure-based antibody engineering, including affinity maturation, humanization, and the generation of antibody-drug conjugates (ADCs).
- NMR higher-order structure (HOS) analysis: Assessing the comparability of a biosimilar antibody and its reference product for biologics development and regulatory purposes.
Contact us for a discussion of your project!
FastLane Proteins for Antibody-Antigen Complex Crystallization & Epitope Mapping
X-ray crystallography and cryo-electron microscopy (cryo-EM) are the gold standards for high-resolution epitope mapping. Currently, the Structural Antibody Database (SabDab) houses over 10,000 antibody structures and more than 9,000 tertiary structures of antibody-antigen complexes at near-atomic resolution. Of these, 6,438 structures were determined using X-ray crystallography, 3,659 using Cryo-Electron Microscopy, and 17 by NMR spectroscopy. These three methods provide the most accurate high-resolution structural insights into antibody-antigen interactions, epitope and paratope mapping. SARomics Biostructures has extensive
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 reveals that Glenzocimab binds to the dimerization site in the D2 domain of GPVI, inhibiting 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 Epitope Mapping?
Commonly used techniques for epitope mapping include the peptide-based hydrogen-deuterium exchange mass spectrometry (HDX-MS), cross-linking mass spectrometry (XL-MS), fast photochemical oxidation of proteins (FPOP), and the more general methods, such as computational methods, X-ray crystallography, and cryo-electron microscopy (cryo-EM). Each of these methods has its own advantages and disadvantages.
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. For mapping the latter group of epitopes, X-ray crystallography and cryo-EM are considered the gold standard due to their ability to provide high-resolution insights into intermolecular interactions. Needless to say, this high level of accuracy significantly enhances antibody targeting. The SARomics Biostructures team possesses proven expertise in the field of antibody-antigen crystallization and has contributed to numerous published high-resolution X-ray structures of antibody-antigen complexes. Contact us to discuss the details of your project!
Entrusting SARomics Biostructures with Your Antibody-Antigen Complex Structure and Epitope Mapping
SARomics Biostructures CRO team has contributed to numerous crystallizations, X-ray structure determinations, and epitope mappings of 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; additional papers can be found in the PDF on the right.
- 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.
- Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies. Ferrari (Autolus Therapeutics) et al., 2024, Nat Commun, 15.
- SRF388 Fab in complex with IL-27. Skladanowska (Surface Oncology Inc.) et al., 2022. Cell Reports, 41, 111490.
- Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity. Liu (Adimab, LLC) et al., 2023, MAbs, 15, 2189974-21899.
- Structures of activin ligand traps using natural sets of type I and type II TGFβ receptors. Goebel (Acceleron Pharma) et al., 2022. iScience, 25, 103590.
- The bispecific 4-1BB x 5T4 agonist, ALG.APV-527 mediates strong T-cell activation and potent anti-tumor activity. Nelson (Alligator Bioscience AB) et al., 2022, Mol. Cancer Ther., 22-0395.
- Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition. Billiald (Acticor Biotech) et al., 2022. Blood Adv (2023) 7 (7): 1258–1268.
- DutaFab in complex with its antigens PDGF and VEGFA. Beckmann (Roche) et al., 2021. Nat Comm, 12:708.
- Bispecific Antibody Mediating PD-L1–Dependent CD28 Co-stimulation on T Cells for Enhanced Tumor Control. Majocchi (Light Chain Bioscience – Novimmune SA) et al., 2024. Cancer Immunol Res, 0298.
NMR Higher-Order Structure (HOS) Analysis of Biosimilars
Higher-order structure analysis of biosimilars and their comparability is critical to developing new biologics that adhere to patient safety principles (see our blog post on the subject). In the blog article, we show the superiority of the NMR-based approach, which provides information at the atomic level, compared to other methods used for higher-order structure analysis.
At SARomics Biostructures, we use advanced NMR spectroscopy to analyze higher-order structures of antibodies. We acquire a unique fingerprint of the 3D conformation of large, complex molecules, such as biologics. For biosimilar structure comparability analysis, 2D 13C NMR spectra of the methyl region of the biomolecules are acquired, and the biosimilar and originator spectra are compared. The method is based on natural abundance and does not require additional expensive labeling. By matching the NMR fingerprint of a given protein to its high-resolution 3D structure, determined, e.g., by X-ray crystallography, or to a fingerprint of another protein batch of the therapeutic monoclonal antibody or its biosimilar, we can rapidly analyse the higher order structure, assess its comparability and show that the a biosimilar and its originator, or different batches or alternative preparations of the same biologic, have identical higher order structures. Additionally, biomolecules can be studied in the formulation buffer to optimize formulations and compare batches. Depending on the biomolecule, approximately 1-2 mg of protein is sufficient for HOS analysis.
The presentation below provides details of the method and a case study.
Featured Publication: The Structure of Bispecific Antibodies Driven by Unique Light Chains.
P Malinge, X Chauchet, J Bourguignon, N Bosson, S Calloud, T Bautzova, M Borlet, M Laursen, V Kelpsas, N Rose, F Gueneau, U Ravn, G Magistrelli & N Fischer (2024).
Structural analysis of light chain-driven bispecific antibodies targeting CD47 and PD-L1. mAbs 16, NO. 1, 2362432.
https://doi.org/10.1080/19420862.2024.2362432
The authors developed a bispecific antibody format that, unlike natural antibodies, which mainly rely on the heavy chain (HC), drives antigen binding and specificity through the light chain (LC). To better understand epitope-paratope interactions in the antibody-antigen complex, the SARomics team determined the X-ray crystallographic structures of an antigen-binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1 (found in our FastLane™ Premium library). Structure analysis revealed the dominant contribution of the light chain, demonstrating that it can also mediate high-affinity binding, enabling the creation of bispecific antibodies with native structures and thereby enhancing their therapeutic potential. (PDB IDs: 8RP8 and 8RPB).
We invite you to visit our publications page for additional examples of projects we have contributed to.