OX40 (CD134) is a potent costimulatory molecule found on the surface of activated CD4(+) and CD8(+) T cells. Immunotherapy with OX40 agonists administered in vivo has demonstrated efficacy in several murine tumor models. A phase I clinical trial is currently underway in patients with advanced cancer using a mouse anti-CD134 monoclonal antibody. Therapy with this antibody will likely be limited to one cycle because patients develop neutralizing human anti-mouse antibody (HAMA). Therefore, we developed a humanized OX40 agonist that links the extracellular domain of human OX40L to the Fc domain of human IgG(1) via a trimerizing isoleucine zipper domain (ILZ). Physical characterization by velocity sedimentation revealed that this novel construct, hFcILZOX40L, was assembled into hexamers in which the Fc domains formed three disulfide-bonded dimers and the ILZ-OX40L domains formed two trimers. Trimerization of the ILZ domain was necessary to achieve appropriate assembly. In vitro biologic activity of the hFcILZOX40L hexamer was equivalent to the activity of agonist antibodies in plate-bound assays and was superior when the agonists were tested as soluble agents. Our ultimate goal is to use this recombinant molecule in a future clinical trial, and we feel that the OX40L hexamer will have equivalent or superior agonist activity in vivo when compared to an anti-OX40 antibody.