Recombinant Antibody Fusion Proteins for Depletion of Autoreactive B Lymphocytes

For most autoimmune diseases known, treatment options are very limited. In fact, there is not a single autoimmune indication where a disease-specific treatment regimen has been described. It was the aim of this work to investigate novel strategies for specific depletion of autoreactive B cells as the root cause of many autoimmune diseases. Taken together, this work shows that single-chain fusion proteins can be engineered to target B cells specific for the autoantigen MOG for depletion via immune-effector mechanisms. This was achieved by coupling the MOG extracellular domain to a C-terminally attached effector domain consisting of either an Fc part of human IgG1 or a single-chain variable domain (scFv) directed against CD3e. These proteins induced the efficient depletion of MOG-specific B cells in vitro, ex vivo and in vivo, and thus represent a novel approach for recombinant proteins targeting autoreactive B cells in antibody-mediated autoimmunity. Due to the exacerbating potential of the autoantigen fragment, their use as a therapeutic option in autoimmune conditions requires further development.For most autoimmune diseases known, treatment options are very limited. In fact, there is not a single autoimmune indication where a disease-specific treatment regimen has been described. It was the aim of this work to investigate novel strategies for specific depletion of autoreactive B cells as the root cause of many autoimmune diseases. Taken together, this work shows that single-chain fusion proteins can be engineered to target B cells specific for the autoantigen MOG for depletion via immune-effector mechanisms. This was achieved by coupling the MOG extracellular domain to a C-terminally attached effector domain consisting of either an Fc part of human IgG1 or a single-chain variable domain (scFv) directed against CD3e. These proteins induced the efficient depletion of MOG-specific B cells in vitro, ex vivo and in vivo, and thus represent a novel approach for recombinant proteins targeting autoreactive B cells in antibody-mediated autoimmunity. Due to the exacerbating potential of the autoantigen fragment, their use as a therapeutic option in autoimmune conditions requires further development

Impact of Tryptophan Oxidation in Complementarity-Determining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance

This work is licensed under a Creative Commons Attribution 4.0 International License.Purpose Tryptophan’s (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. Methods Selective Trp oxidation was induced by 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. Results Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. Conclusions Trp oxidation in CDRs can adversely impact mAbs’ conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product