Abstract
Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another’s binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen’s unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties.
Highlights
High-throughput epitope binning experiments on real-time label-free biosensors are commonly used in early stage discovery of therapeutic monoclonal antibodies to sort large panels of mAbs into epitope families or “bins” based upon their ability to block one another’s binding to their specific antigen in a pairwise and combinatorial fashion
By characterizing four panels of mAbs targeting unrelated protein antigens —namely human proprotein convertase subtilisin/kexin type 9 (PCSK9), human progranulin (PGRN), human epidermal growth factor receptor (EGFR), and hen egg white lysozyme (HEL) —we find that displacements appear to occur on any antigen, if the mAb panel contains appropriate epitope diversity
To investigate the mechanism of mAb displacement, we first focus on two mAbs that bind with high specificity and high affinity towards PCSK9, namely mAbs C34 and 69, showing apparent KD values in the single digit picomolar range when analyzed by surface plasmon resonance (SPR) biosensors (Fig 2A)
Summary
High-throughput epitope binning experiments on real-time label-free biosensors are commonly used in early stage discovery of therapeutic monoclonal antibodies (mAbs) to sort large panels of mAbs into epitope families or “bins” based upon their ability to block one another’s binding to their specific antigen in a pairwise and combinatorial fashion. Biosensor Study of Antibody Displacement specific roles of these authors are articulated in the ‘author contributions’ section. Three of these co-authors, WM, AR and PS, were employed by Pfizer during the study but at the submission date were employed at other commercial companies, namely Bristol-Myers Squibb (WM and PS) and Compugen USA Inc. These companies did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript
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