Journal Article

Generation and characterization of OX40-ligand fusion protein that agonizes OX40 on T-Lymphocytes

Nagai, Hodaka, Suzuki, Ayano, So, Takanori, Ishii, Naoto, Sato, Ayaka, Shibui, Nagito, Morita, Masashi, Hikosaka-Kuniishi, Mari, Matsuyama, Shimpei, Ito, Aya

2024

Overview

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed on the surface of activated T cells. Upon interaction with its cognate ligand, OX40L, OX40 transmits costimulatory signals to antigen-primed T cells, promoting their activation, differentiation, and survival — processes essential for the establishment of adaptive immunity. Although the OX40-OX40L interaction has been extensively studied in the context of disease treatment, developing a substitute for the naturally expressed membrane-bound OX40L, particularly a multimerized OX40L trimers, that effectively regulates OX40-driven T cell responses remains a significant challenge. In this study, we successfully engineered soluble OX40L-fusion proteins capable of robustly activating OX40 on T cells. This was achieved by incorporating functional multimerization domains into the TNF homology domain of OX40L. These OX40L proteins bound to OX40, subsequently activated NF-κB signaling, and induced cytokine production by T cells in vitro . In vivo , mice treated with one of the OX40L-fusion proteins — comprising a single-chain OX40L trimer linked to the C-terminus of the human IgG1 Fc domain, forming a dimer of trimers — exhibited significantly enhanced clonal expansion of antigen-specific CD4 + T cells during the primary phase of the immune response. A comparable antibody-fusion single-chain TNF protein incorporating 4-1BBL, CD70 (CD27L), or GITRL in place of OX40L elicited similar in vivo T cell responses. Thus, we propose that optimizing the multimerization of OX40L proteins through innovative design strategies may facilitate the development of more effective agonists for targeted immunotherapies.

• Publisher: Frontiers Media SA,Frontiers Media S.A
• Subject: Adaptive immunity / Agonists / Animals / Antibodies / Antigens / C-Terminus / CD4 antigen / CD70 antigen / Cell activation / Cell differentiation / Cloning / co-stimulation / Cytokines / Dihydrofolate reductase / Fusion protein / Gene amplification / Homology / Humans / Immunoglobulin G / Immunotherapy / Ligands / Lymphocyte Activation - immunology / Lymphocytes / Lymphocytes T / Mice / Mice, Inbred C57BL / NF-κB protein / OX40 / OX40 Ligand - genetics / OX40 Ligand - immunology / OX40 Ligand - metabolism / OX40L / Ox40L protein / Plasmids / Proteins / Receptors, OX40 - agonists / Receptors, OX40 - immunology / Receptors, OX40 - metabolism / Recombinant Fusion Proteins - genetics / Recombinant Fusion Proteins - immunology / Recombinant Fusion Proteins - pharmacology / Signal Transduction / T cell / T-Lymphocytes - immunology / T-Lymphocytes - metabolism / TNF receptor superfamily / TNF superfamily / Trimers / Tumor necrosis factor / Tumor necrosis factor-TNF