Leveraging Cross-Linking Mass Spectrometry for Modeling Antibody-Antigen Complexes.

Elucidating antibody-antigen complexes at the atomic level is of utmost interest for understanding immune responses and designing better therapies. Cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for mapping protein-protein interactions, suggesting valuable structural insights. However, the use of XL-MS studies to enable epitope/paratope mapping of antibody-antigen complexes is still limited up to now. XL-MS data can be used to drive integrative modeling of antibody-antigen complexes, where cross-links information serves as distance restraints for the precise determination of binding interfaces. In this approach, XL-MS data are employed to identify connections between binding interfaces of the antibody and the antigen, thus informing molecular modeling. Current literature provides minimal input about the impact of XL-MS data on the integrative modeling of antibody-antigen complexes. Here, we applied XL-MS to retrieve information about binding interfaces of three antibody-antigen complexes. We leveraged XL-MS data to perform integrative modeling using HADDOCK (active-passive residues and distance restraints strategies) and AlphaLink2. We then compared these three approaches with initial predictions of investigated antibody-antigen complexes by AlphaFold Multimer. This work emphasizes the importance of cross-linking data in resolving conformational dynamics of antibody-antigen complexes, ultimately enhancing the design of better protein therapeutics and vaccines.

Preclinical risk assessment strategy to mitigate the T-cell dependent immunogenicity of protein biotherapeutics: State of the art, challenges and future perspectives.

Protein therapeutics hold a prominent role and have brought significant diversity in efficacious medicinal products. Not just monoclonal antibodies and different antibody formats (pegylated antigen-binding fragments, bispecifics, antibody-drug conjugates, single chain variable fragments, nanobodies, dia-, tria- and tetrabodies), but also purified blood products, growth factors, recombinant cytokines, enzyme replacement factors, fusion proteins are all good instances of therapeutic proteins that have been developed in the past decades and approved for their value in oncology, immune-oncology, and autoimmune diseases discovery programs. Although there was an ingrained belief that fully humanized proteins were expected to have limited immunogenicity, adverse effects associated with immune responses to biological therapies raised some concern in biotech companies. Consequently, drug developers are designing strategies to assess potential immune responses to protein therapeutics during both the preclinical and clinical phases of development. Despite the many factors that can contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) immunogenicity seems to play a crucial role in the development of anti-drug antibodies (ADAs) to biologics. A broad range of methodologies to predict and rationally assess Td immune responses to protein drugs has been developed. This review aims to briefly summarize the preclinical immunogenicity risk assessment strategy to mitigate the risk of potential immunogenic candidates coming towards clinical phases, discussing the advantages and limitations of these technologies, and suggesting a rational approach for assessing and mitigating Td immunogenicity.

Development of a Liquid Chromatography and High-Resolution and -Accuracy Mass Spectrometry Method to Evaluate New Biotherapeutic Entity Processing in Human Liver Lysosomes.

Biotherapeutic immunogenicity remains a great challenge for researchers because multiple factors trigger immune responses. Predicting and assessing the potential human immune response against biological drugs could represent an impressive breakthrough toward generating potentially safer and more efficacious therapeutic proteins. This article describes an in vitro assay that can contribute to evaluating the potential immunogenicity of biotherapeutics by focusing on lysosomal proteolysis. We selected human liver lysosomes (hLLs) from four different donors as a surrogate in vitro model instead of APC lysosomes because they are a ready-to-use lysosomal source. To assess the biological comparability of this surrogate to APC lysosomal extract, we compared the proteome content of hLLs with literature data of lysosomal fractions extracted from murine bone marrow and human blood-derived dendritic cells. Then we tested infliximab (IFX; Remicade) under different proteolytic conditions using liquid chromatography and high-resolution and -accuracy mass spectrometry to better define the degradation kinetics inside the lysosomes. hLLs revealed similar enzymatic content compared with human and murine dendritic cell lysosomes. Degradation assays demonstrated that our liquid chromatography and high-resolution and -accuracy mass spectrometry method could identify both the intact protein and the peptides resulting from proteolysis with high specificity and resolution. The rapid and easy assay described in this article can be extremely useful for evaluating the immunogenic risk associated with therapeutic proteins. In addition, this method can complement information from MHC class II-associated peptide proteomics assays and other in vitro and in silico techniques.