Decorating Antigen with Multivalent CD22 Ligands to Prevent B-cell Mediated Anti-Drug Antibody Formation

Abstract

Over the past few decades, the field of drug discovery has slowly been shifting towards the development of biotherapeutics. They offer many benefits over traditional small molecules imparted by their high specificity such as low off-target effects and low chance of drug-drug interactions. However, because these are large and complex biomolecules, anti-drug antibodies (ADAs) can easily be formed reducing their efficacy and causing moderate to severe side effects. As B-cells are the cells responsible for antibody production, inhibiting their activation may supress or completely forgo ADA formation. To achieve this goal, we opted to link high affinity murine CD22 (mCD22) ligands to the model antigen Ovalbumin (OVA), to examine if costimulatory binding of the inhibitory CD22 receptor can prevent OVA specific antibody formation. A novel synthetic route was developed to synthesize large quantities of a previously reported high affinity mCD22 ligand and novel di- and tetravalent scaffolds were developed based on existing literature and used to multimerize this mCD22 ligand. Microscale thermophoresis showed sub-micromolar binding of these di- and tetravalent ligands to mCD22. The conjugation of these multivalent ligands to OVA was optimized and it was shown to lead to highly increased uptake of the conjugates by splenic B-cells as seen by FACS. In vivo, the OVA modified with the di- and tetravalent ligand showed an over ten-fold reduction in anti-OVA IgG1 production. Unfortunately, the total antibody production against the OVA-conjugates, was not changed. This observation likely indicates that antibodies are generated against the linker or ligand, suggesting that this way of presentation does not (fully) supress B-cell activation. The possible reasons as to why this was observed are discussed, and suggestions are made for further research.