Development and characterization of recombinant human Fc:OX40L fusion protein linked via a coiled-coil trimerization domain

Abstract

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 disulfidebonded 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.

The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer

“…The anti-tumor activity of anti-mouse CD40 in these studies is lost upon introduction of the D265A mutation into its Fc domain [13], which results in an antibody with no measurable FcγR binding [6]. It has also been shown that the in vitro activity of antibodies directed against other TNFRs in human cells, such as OX40, GITR, CD137 and CD27, requires, or is greatly enhanced by FcγR engagement or immobilisation [25,26]. While the evidence suggests that a requirement for cross linking is a common property of antibodies targeting TNFRs receptors, there are some exceptions.…”

OX40, OX40L and Autoimmunity: a Comprehensive Review

“…Such problems have been reported in the development of agonistic OX40L fusion protein [145]. However, such antibodies were not generated in a trial of OX40L blockade [142].…”

Immunosilencing a Highly Immunogenic Protein Trimerization Domain

“…These include cancer therapeutics that have been tested in clinical trials, such as the TNF superfamily member CD40 ligand (6 -8), as well as therapeutics that have been tested preclinically, such as OX40 ligand (9) and TRAIL (10). Experimental protein vaccines, some, which are considered for clinical trials, also exploit IZ and/or Fd and include the spike proteins of human immunodeficiency virus (HIV-1) (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), respiratory syncytial virus (21,22), and influenza virus (4,(23)(24)(25)(26)(27)(28).…”