A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

Abstract

Solution-based affinity assays are used for the selection and characterization of proteins that could be developed into therapeutic molecules. However, these assays have limitations for cell-surface proteins as in most cases their purification requires detergent solubilization and are unlikely to assume conformations in solution that resemble their native states in cell membranes. This report describes a novel electrochemiluminescence-based method, called MSD-CAT, for the affinity analysis of antibodies binding to cell-surface receptors. MSD-CAT was used to evaluate the binding of monoclonal antibodies, Fab fragments, and bispecific antibodies targeting the cell-surface receptor interleukin 3 receptor alpha (CD123) and the results were compared to data obtained using surface plasmon resonance (SPR). The data showed that MSD-CAT can be successfully applied to determine binding affinity on cells in a label free format and without the need for laborious solubilization procedures to generate recombinant antigen. In addition, this method has the potential for high-throughput application while enabling simultaneous determination of equilibrium dissociation constant (K_D) and receptor density within the same experiment.

Keywords: Affinity; Bispecific antibodies; Cell affinity; MSD; MSD-CAT; SPR.

Fig. 1

Representative SPR sensorgrams obtained by Biacore of antibody mAb I3RB18 Fab (I3RB119), and BsAbs binding to recombinant human antigen CD123 SP1 (A) and its truncated variant CD123 SP2 (B).

Fig. 2

Schematic representation of the MSD-CAT procedure. Antibody of interest at a constant concentration (1) is mixed with a serial dilution of cells expressing the antigen (2). The reaction mixture is incubated to form the Ab-receptor complex and to allow the reaction to reach equilibrium (3). Following incubation and complex formation, the reaction mixture is centrifuged to separate antibody bound to cells from free antibody in supernatant (4). The supernatant containing free antibody is added to a streptavidin-MSD plate in which the capture reagent has been previously immobilized (5). Free antibody in the supernatant binds to the capture reagent in the plate (6) and it is detected using Ruthenylated anti-IgG (7). Luminescence signal is measured, and the results processed as described in the MSD-CAT data analysis section to determine affinity.

Fig. 3

Representative binding curves for MSD-CAT affinity analysis for cell surface expressed CD123, SP1 (A) and CD123 SP2 (B). The Y-axis shows % free anti-receptor molecules (BsAb, Fab or mAb) or CD123 negative control mAb (I3CB15) while the X-axis shows the concentration of cell surface antigen. Each curve represents a different but fixed concentration of mAb, bispecific or Fab as indicated.

Fig. 4

Binding curves for MSD-CAT affinity analysis for cyno CD123. The Y-axis shows % free anti-receptor molecules (mAb or its Fab) while the X-axis shows the concentration of cell surface antigen. Each curve represents a different but fixed concentration of mAb, or Fab as indicated. The figure also shows a plot of the control commercial antibody, 7G3, showing cross reactivity to human CD123 (SP1).