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

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: The novel bispecific KA-5484 binds both OX40L and TNFα with high affinity.Dual targeting of OX40L and TNFα enables synergistic immune modulation.KA-5484 outperforms control antibodies in binding and inflammatory assays.KA-5484 shows superior efficacy in mitigating arthritis in the collagen antibody-induced arthritis mouse model.Enhanced cross-species reactivity supports the translational relevance of KA-5484.