The determination and correlation of molecular and cellular equilibrium Kd and kinetic koff values for small molecule allosteric antagonists of LFA-1

Abstract Molecular K d and k off parameters are often used to define the molecular potency of drugs. These constants, however, are determined on purified target proteins, and their relationship to in vivo binding phenomena is poorly understood. Herein, we report two novel assays to determine the off-rates of allosteric antagonists from lymphocyte function-associated antigen 1 (LFA-1). The SPR assay involves using the non-blocking mAb TS2/4 to immobilize full-length LFA-1 on a hydrophilic chip surface, and the soluble, native ligand sICAM-1 to probe the fraction of free LFA-1. To determine the fraction of free LFA-1 on cell surfaces, a flow cytometry assay was developed utilizing the fluorophore-labeled Fab R3.1. The R3.1 antibody has been previously demonstrated to block the ability of both ICAM-1 and antagonists to bind to purified and cell-surface LFA-1. The molecular and ex vivo cellular parameters were determined for a set of nine structurally-related LFA-1 allosteric antagonists. The relationships between the parameters determined in the ELISA ( K d ), SPR ( k off ), and flow cytometry ( k off ) assays were shown to be linear with slopes approximately equal to 1, and a correlation analysis showed that the three assay datasets were equivalent at the α  = 0.05 level. These results were unexpected, as the ELISA and SPR assays involve high affinity LFA-1, and the flow cytometry assays involve cell surface LFA-1 in whole-blood, in which a distribution of affinity states would be expected. Nevertheless, the results presented herein show that the K d and k off 's determined in molecular assays can be used as predictors of LFA-1 receptor occupancy in ex vivo assays.