OX40 Ligand-Mannose-Binding Lectin Fusion Protein Induces Potent OX40 Cosignaling in CD4⁺ T Cells

Abstract

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is induced on activated T cells. Membrane-bound OX40 ligand (OX40L) expressed by activated antigen-presenting cells induces OX40 signaling, which promotes T cell immunity. OX40 agonism would be a potential target for immunotherapy, however, it remains unclear how the activity of OX40 can be successfully controlled by a designer OX40L protein. We prepared a soluble OX40L protein possessing a PA-peptide tag and a collagenous trimerization domain from mannose-binding lectin (MBL), and tested whether PA-MBL-OX40L fusion protein worked as an agonist for OX40. We found that the majority of recombinant PA-MBL-OX40L protein purified from culture supernatants displayed a trimer structure and bound to cell surface OX40 or OX40-Fc fusion protein in a dose-dependent manner. Upon stimulation of CD4 T cells with TCR/CD3 without CD28, PA-MBL-OX40L displayed significantly increased proliferative and cytokine responses when compared with a benchmark agonistic monoclonal antibody for OX40. Both soluble and immobilized forms of PA-MBL-OX40L induced potent OX40 signaling in CD4 T cells. Mice administered with PA-MBL-OX40L displayed significantly augmented T cell-mediated delayed-type hypersensitivity responses. Our results suggest that activity of OX40L could be engineered to elicit better T cell responses by rational design of its assembly and architecture.

Fundamental Characterization of Antibody Fusion-Single-Chain TNF Recombinant Proteins Directed against Costimulatory TNF Receptors Expressed by T-Lymphocytes

“…A pREP4 vector (Invitrogen, Waltham, MA, USA) containing a cDNA encoding mouse Ox40l ( Tnfsf4 ) was previously described [ 36 ]. A qCR TM -Blunt II-TOPO TM vector (#450245, Thermo Fisher, Waltham, MA, USA) containing a cDNA encoding mouse Cd70 or mouse 41bb ( Tnfrsf9 ) was previously described [ 37 ]. PCR primers were purchased from Fasmac Co., Ltd. (Kanagawa, Japan).…”

“…A plasmid encoding mouse OX40 extracellular region ( 21 Thr- 211 Pro) and human IgG1-Fc (mouse OX40-Fc) in the pEF-Fc vector [ 41 ], and a plasmid encoding mouse 4-1BB extracellular region ( 24 Val− 187 Leu) and human IgG1-Fc (mouse 4-1BB-Fc) in the pEF-Fc vector [ 37 ] were previously described. A plasmid encoding mouse CD27 extracellular region ( 24 Pro− 182 Arg) and human IgG1-Fc (mouse CD27-Fc) in the pEF-Fc vector was constructed in this study, and a PCR fragment (gac tac aag gat gac gat gac aag ctc gat gga gga tac cca tac gat gtt cca gat tac gct) corresponding to a Flag−HA tag peptide (DYKDDDDKLDGGYPYDVPDYA) was inserted into the 3′ end of the CD27−Fc gene by In-Fusion cloning.…”

“…Delayed-type hypersensitivity (DTH) response was evaluated as previously described [ 37 , 42 ]. C57BL/6 mice from both male and female mice aged between 12 and 14 weeks were immunized subcutaneously at the tail with 200 µL of 1.25 mg/mL methyl BSA (mBSA) (A1009, Merck) emulsified with complete Freund’s adjuvant (CFA) (F5881, Merck) on day 0.…”