{"title":"一种针对TNF-α和IL-6受体的新型双特异性抗体作为一种有效的炎症免疫治疗剂。","authors":"Mansooreh Heravi, Shadi Damough, Ladan Mafakher, Yeganeh Talebkhan, Leila Nematollahi, Leila Ghazizadeh, Soheila Ajdari","doi":"10.1007/s00253-025-13596-x","DOIUrl":null,"url":null,"abstract":"<p>Tumor necrosis factor-alpha and interleukin-6 are proinflammatory cytokines involved in several autoimmune diseases, including rheumatoid arthritis. Although monoclonal antibodies targeting these cytokines or their receptors are successful treatment approaches for autoimmune diseases, approximately 30% of treated patients fail to respond. Therefore, the designation of more effective and versatile therapeutics for simultaneously targeting multiple inflammatory pathways is pivotal. This study aimed to design a recombinant bispecific antibody (BisAb) targeting TNF-α and IL-6 receptor based on the complementarity-determining regions (CDRs) of adalimumab and tocilizumab antibodies. The physicochemical properties and tertiary structure of the modeled bispecific antibody proteins were studied through bioinformatics. The interaction of the designed bispecific antibody with human TNF-α and IL-6R was computed by molecular docking and molecular dynamics. The recombinant BisAb (60kDa) was expressed in <i>Escherichia coli.</i> The ELISA results demonstrated that the affinity of BisAb and its Ada-scFv moiety to TNF-α was quite similar with <i>K</i><sub>aff</sub> values of 7.7 and 9.4 × 10<sup>–13</sup> M, respectively. The recombinant BisAb and Toci-scFv also represented similar affinities towards IL-6R (<i>K</i><sub>aff</sub> values of 1.65 and 1.87 × 10<sup>–12</sup> M), closely comparable with the parental tocilizumab antibody. A dose-dependent neutralization of TNF-mediated cytotoxicity was also observed on L929 cells. Decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3) was determined through western blotting with specific antibodies, indicating the blockade of IL-6 receptors before the preincubation with IL-6. The designed BisAb, a minimized biotherapeutic molecule, could successfully target two main ligands (IL-6R and TNF-α) involved in rheumatoid arthritis and many inflammatory/infectious diseases.</p><p>• <i>A new bispecific antibody was designed to target TNF-α and IL-6R.</i></p><p>• <i>In silico studies confirmed the physicochemical and structural properties of BisAb.</i></p><p>• <i>The best-modeled protein was recombinantly expressed in Escherichia coli.</i></p><p>• <i>BisAb significantly inhibited TNF-α-mediated cytotoxicity.</i></p><p>• <i>BisAb could efficiently suppress the IL-6-related phosphorylation of STAT3 protein.</i></p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":4.3000,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13596-x.pdf","citationCount":"0","resultStr":"{\"title\":\"A novel bispecific antibody targeting TNF-α and IL-6 receptor as a potent immunotherapeutic agent for inflammation\",\"authors\":\"Mansooreh Heravi, Shadi Damough, Ladan Mafakher, Yeganeh Talebkhan, Leila Nematollahi, Leila Ghazizadeh, Soheila Ajdari\",\"doi\":\"10.1007/s00253-025-13596-x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Tumor necrosis factor-alpha and interleukin-6 are proinflammatory cytokines involved in several autoimmune diseases, including rheumatoid arthritis. Although monoclonal antibodies targeting these cytokines or their receptors are successful treatment approaches for autoimmune diseases, approximately 30% of treated patients fail to respond. Therefore, the designation of more effective and versatile therapeutics for simultaneously targeting multiple inflammatory pathways is pivotal. This study aimed to design a recombinant bispecific antibody (BisAb) targeting TNF-α and IL-6 receptor based on the complementarity-determining regions (CDRs) of adalimumab and tocilizumab antibodies. The physicochemical properties and tertiary structure of the modeled bispecific antibody proteins were studied through bioinformatics. The interaction of the designed bispecific antibody with human TNF-α and IL-6R was computed by molecular docking and molecular dynamics. The recombinant BisAb (60kDa) was expressed in <i>Escherichia coli.</i> The ELISA results demonstrated that the affinity of BisAb and its Ada-scFv moiety to TNF-α was quite similar with <i>K</i><sub>aff</sub> values of 7.7 and 9.4 × 10<sup>–13</sup> M, respectively. The recombinant BisAb and Toci-scFv also represented similar affinities towards IL-6R (<i>K</i><sub>aff</sub> values of 1.65 and 1.87 × 10<sup>–12</sup> M), closely comparable with the parental tocilizumab antibody. A dose-dependent neutralization of TNF-mediated cytotoxicity was also observed on L929 cells. Decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3) was determined through western blotting with specific antibodies, indicating the blockade of IL-6 receptors before the preincubation with IL-6. The designed BisAb, a minimized biotherapeutic molecule, could successfully target two main ligands (IL-6R and TNF-α) involved in rheumatoid arthritis and many inflammatory/infectious diseases.</p><p>• <i>A new bispecific antibody was designed to target TNF-α and IL-6R.</i></p><p>• <i>In silico studies confirmed the physicochemical and structural properties of BisAb.</i></p><p>• <i>The best-modeled protein was recombinantly expressed in Escherichia coli.</i></p><p>• <i>BisAb significantly inhibited TNF-α-mediated cytotoxicity.</i></p><p>• <i>BisAb could efficiently suppress the IL-6-related phosphorylation of STAT3 protein.</i></p>\",\"PeriodicalId\":8342,\"journal\":{\"name\":\"Applied Microbiology and Biotechnology\",\"volume\":\"109 1\",\"pages\":\"\"},\"PeriodicalIF\":4.3000,\"publicationDate\":\"2025-10-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://link.springer.com/content/pdf/10.1007/s00253-025-13596-x.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Applied Microbiology and Biotechnology\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s00253-025-13596-x\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Applied Microbiology and Biotechnology","FirstCategoryId":"5","ListUrlMain":"https://link.springer.com/article/10.1007/s00253-025-13596-x","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
A novel bispecific antibody targeting TNF-α and IL-6 receptor as a potent immunotherapeutic agent for inflammation
Tumor necrosis factor-alpha and interleukin-6 are proinflammatory cytokines involved in several autoimmune diseases, including rheumatoid arthritis. Although monoclonal antibodies targeting these cytokines or their receptors are successful treatment approaches for autoimmune diseases, approximately 30% of treated patients fail to respond. Therefore, the designation of more effective and versatile therapeutics for simultaneously targeting multiple inflammatory pathways is pivotal. This study aimed to design a recombinant bispecific antibody (BisAb) targeting TNF-α and IL-6 receptor based on the complementarity-determining regions (CDRs) of adalimumab and tocilizumab antibodies. The physicochemical properties and tertiary structure of the modeled bispecific antibody proteins were studied through bioinformatics. The interaction of the designed bispecific antibody with human TNF-α and IL-6R was computed by molecular docking and molecular dynamics. The recombinant BisAb (60kDa) was expressed in Escherichia coli. The ELISA results demonstrated that the affinity of BisAb and its Ada-scFv moiety to TNF-α was quite similar with Kaff values of 7.7 and 9.4 × 10–13 M, respectively. The recombinant BisAb and Toci-scFv also represented similar affinities towards IL-6R (Kaff values of 1.65 and 1.87 × 10–12 M), closely comparable with the parental tocilizumab antibody. A dose-dependent neutralization of TNF-mediated cytotoxicity was also observed on L929 cells. Decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3) was determined through western blotting with specific antibodies, indicating the blockade of IL-6 receptors before the preincubation with IL-6. The designed BisAb, a minimized biotherapeutic molecule, could successfully target two main ligands (IL-6R and TNF-α) involved in rheumatoid arthritis and many inflammatory/infectious diseases.
• A new bispecific antibody was designed to target TNF-α and IL-6R.
• In silico studies confirmed the physicochemical and structural properties of BisAb.
• The best-modeled protein was recombinantly expressed in Escherichia coli.
期刊介绍：
Applied Microbiology and Biotechnology focusses on prokaryotic or eukaryotic cells, relevant enzymes and proteins; applied genetics and molecular biotechnology; genomics and proteomics; applied microbial and cell physiology; environmental biotechnology; process and products and more. The journal welcomes full-length papers and mini-reviews of new and emerging products, processes and technologies.
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