{"title":"A novel bispecific antibody targeting OX40L and TNFα for the targeted treatment of rheumatoid arthritis.","authors":"Qun Zhang, Shuliang Song","doi":"10.1093/abt/tbag003","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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. <i>In vivo</i> efficacy was tested in a collagen antibody-induced arthritis (CAIA) mouse model.</p><p><strong>Results: </strong>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<sup>-10</sup> 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.</p><p><strong>Conclusion: </strong>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.</p><p><strong>Highlights: </strong>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.</p>","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":"9 1","pages":"76-85"},"PeriodicalIF":4.8000,"publicationDate":"2026-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12967327/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Antibody Therapeutics","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/abt/tbag003","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2026/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"Medicine","Score":null,"Total":0}
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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: 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.
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