Profiling antibody polyreactivity and polyspecificity using HT-SPR on the Carterra^® LSA^®

Key Takeaways

• Monoclonal antibodies are an important class of therapeutic proteins used to treat a wide range of diseases
• Having a better understanding of the polyreactivity and polyspecificity of potential monoclonal antibody candidates can result in more effective and efficient development of therapeutics
• The LSA, which runs HT-SPR, can be used to fully automate large scale, real-time screening of panels of antibodies to gain deep information on their specificity or off-target binding during early development

Introduction

Monoclonal antibodies (mAbs) are one of the most important classes of therapeutic proteins, which are used to treat a wide range of diseases (e.g., oncology, inflammation, and autoimmune diseases). Their use as therapeutics is supported by their ability to bind specifically to their target, have long serum half-lives, and have high tolerability due to the abundance of IgG in the body. Despite these beneficial characteristics, antibodies still regularly fail during therapeutic development. One of the factors causing these failures is linked to polyreactivity and polyspecificity^1-4. Both terms refer to the ability of an antibody to bind to several targets and in the context of drug development to off-target binding. While polyreactivity can be defined as chemical “stickiness,” polyspecificity describes the ability of the antibodies to bind specifically to structurally related epitope on different antigens. Either of these off-target activities can potentially lead to poor pharmacokinetics (PK), potency, bioavailability and/or immunogenicity⁵. Being able to investigate antibody specificity in early discovery is necessary to mitigate costly failure during development phases⁶.

Here, we describe a high-throughput surface plasmon resonance (HT-SPR) assay to profile antibody polyreactivity/polyspecificity. This assay takes advantage of the LSA’s ability to monitor binding interactions of an analyte against 384 ligands in an array. A selection of proteins reflecting the human blood biological environment, proteins displaying specific, biophysical properties (highly glycosylated and low PI) and positive controls were included. In addition recombinant antibody target protein, in this case PD-1, was used to understand polyreactivity/polyspecificity. Subsequently, a fully automated assay enabling the characterization of a panel of antibodies in a single run was carried out. The assay automates several steps including the capture of the protein selections, the injection of the antibodies at two concentrations and the regeneration of the biosensor. The binding levels to the various target proteins were compared to assess the levels of an off-target binding.

Posted by Judicaël Parisot, PhD