A novel bispecific antibody targeting OX40L and TNFα for the targeted treatment of rheumatoid arthritis

Abstract Background Rheumatoid arthritis is driven by T cell dysregulation and TNFα-mediated inflammation. Dual targeting of OX40 ligand (OX40L) and tumor necrosis factor alpha (TNFα) may offer synergistic therapeutic effects. Methods A murine monoclonal antibody against OX40L (KD-0025) was generated and humanized via complementarity-determining region grafting. A bispecific construct incorporating single-chain variable fragments for OX40L and TNFα was expressed in CHO-K1 cells. High-expressing clones were selected, and antibodies were purified using Protein A/G chromatography. Binding to 293 T-OX40L cells, A172 cells, and TNFα was assessed by flow cytometry and ELISA. Functional blockade of OX40L–OX40 and TNFα–TNFR pathways was evaluated using a luciferase reporter assay. Binding kinetics were measured using the ForteBio Octet system. In vivo efficacy was tested in a collagen antibody-induced arthritis (CAIA) mouse model. Results The parental KD-0025 antibody showed EC50 values of 0.4024 μg/ml (human OX40L) and 0.1126 μg/ml (monkey OX40L), with an IC50 of 0.07893 μg/ml. Humanized KA-3966 improved EC50 (0.1374 μg/ml) and had an IC50 of 0.5997 μg/ml. The bispecific antibody KA-5484 demonstrated superior affinity (KD = 1.90 × 10−10 M) and dual-target binding (EC50: 0.1062 and 0.06240 μg/ml for human and monkey OX40L, respectively). It blocked OX40L–OX40 (IC50 = 0.1318 μg/ml) and showed 15–20% greater TNFα pathway suppression than the control antibody KB-1580. KA-5484 significantly reduced inflammation in the CAIA mouse model. Conclusion A humanized bispecific antibody targeting OX40L and TNFα was successfully developed, exhibiting potent dual-target engagement and blockade. The antibody demonstrated synergistic modulation of immune and inflammatory pathways, providing a promising foundation for future preclinical development. Highlights