AFFINITY AND KINETICS OF THE INTERACTION BETWEEN SOLUBLE TRIMERIC OX40 LIGAND, A MEMBER OF THE TUMOR-NECROSIS-FACTOR SUPERFAMILY, AND ITS RECEPTOR OX40 ON ACTIVATED T-CELLS

OX40 ligand (OX40L) and OX40 are members of the tumor necrosis factor and tumor necrosis factor receptor superfamilies, respectively. OX40L is expressed on activated B and T cells and endothelial cell lines, wh ereas OX40 is expressed on activated T cells, A construct for mouse OX 40L was expressed as a soluble protein with domains 3 and 4 of rat CD4 as a tag (sCD4-OX40L). It formed a homotrimer as assessed by chemical cross-linking and gel filtration chromatography, Radio labeled sCD4-O X40L bound to activated mouse T cells with a high affinity (K-D = 0.2- 0.4 nM) and dissociated slowly (k(off) = 4 x 10(-5) s(-1)), The affini ty and kinetics of the OX40L/OX40 interactions were studied using the BIAcore(TM) biosensor, which measures macromolecular interactions in r eal time, The extracellular part of the OX40 antigen was expressed as a soluble monomeric protein and immobilized on the BIAcore sensor chip . sCD4-OX40L bound the OX40 with a high affinity (K-D = 3.8 nM), altho ugh this was lower than that determined on the surface of activated T cells (K-D = 0.2-0.4 nM), where there is likely to be less restriction in mobility of the receptor. In the reverse orientation, sOX40 bound to immobilized sCD4-OX40L with a stoichiometry of 3.1 receptors to one ligand, with low affinity (K-D = 190 nM) and had a relatively fast di ssociation rate constant (k(off) = 2 x 10(-2) s(-1)). Thus if the OX40 receptor is cleaved by proteolysis, it will release any bound ligand and is unlikely to block re-binding of ligand to cell surface OX40 bec ause of the low monomeric affinity.