Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In Vivo
Background: Tetanus toxin, produced by <i>Clostridium tetani</i>, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease. Methods: Herein, we screened thirty-six single variable domains on a heavy...
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2025-06-01
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| Series: | Antibodies |
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| Online Access: | https://www.mdpi.com/2073-4468/14/2/48 |
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| author | Yating Li Kexuan Cheng Jiazheng Guo Yujia Jiang Qinglin Kang Rong Wang Peng Du Chen Gao Yunzhou Yu Zhixin Yang Wei Wang Jiansheng Lu |
| author_facet | Yating Li Kexuan Cheng Jiazheng Guo Yujia Jiang Qinglin Kang Rong Wang Peng Du Chen Gao Yunzhou Yu Zhixin Yang Wei Wang Jiansheng Lu |
| author_sort | Yating Li |
| collection | DOAJ |
| description | Background: Tetanus toxin, produced by <i>Clostridium tetani</i>, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease. Methods: Herein, we screened thirty-six single variable domains on a heavy chain (VHHs) binding to the light chain (L) and the translocation domain (HN) (L-HN) fragment of TeNT from a phage-display library. Then, the L-HN-specific clones were identified, humanized, and fused with a human fragment crystallizable region (hFc) to form humanized VHH-hFc fusion proteins. Results: The humanized VHH-hFc fusion proteins TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc possessed potent efficacy with high binding affinity, specificity, and neutralizing activity. Only 0.3125 μg was required for TL-16-h1-hFc or TL-25-h1-hFc, and 0.625 μg was required for TL-34-h1-hFc to provide full protection against 10 × Lethal Dose 50 (LD<sub>50</sub>) TeNT. In the prophylactic setting, 125 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc provided full protection even when they were injected 12 days before exposure to 10 × LD<sub>50</sub> TeNT, while TL-34-h1-hFc was less effective. In the therapeutic setting, 25 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc could provide complete protection when administered 24 h after exposure to 5 × LD<sub>50</sub> TeNT, while TL-34-h1-hFc required 50 μg/kg. Conclusion: Our results suggest that TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc provide a bright future for the development of anti-TeNT preventive or therapeutic drugs. |
| format | Article |
| id | doaj-art-846da07bd7554c35ad37f08545f2dd22 |
| institution | Kabale University |
| issn | 2073-4468 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibodies |
| spelling | doaj-art-846da07bd7554c35ad37f08545f2dd222025-08-20T03:32:26ZengMDPI AGAntibodies2073-44682025-06-011424810.3390/antib14020048Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In VivoYating Li0Kexuan Cheng1Jiazheng Guo2Yujia Jiang3Qinglin Kang4Rong Wang5Peng Du6Chen Gao7Yunzhou Yu8Zhixin Yang9Wei Wang10Jiansheng Lu11Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaDepartment of Occupational Health and Occupational Diseases, College of Public Health, Zhengzhou University, Zhengzhou 450001, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, ChinaBackground: Tetanus toxin, produced by <i>Clostridium tetani</i>, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease. Methods: Herein, we screened thirty-six single variable domains on a heavy chain (VHHs) binding to the light chain (L) and the translocation domain (HN) (L-HN) fragment of TeNT from a phage-display library. Then, the L-HN-specific clones were identified, humanized, and fused with a human fragment crystallizable region (hFc) to form humanized VHH-hFc fusion proteins. Results: The humanized VHH-hFc fusion proteins TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc possessed potent efficacy with high binding affinity, specificity, and neutralizing activity. Only 0.3125 μg was required for TL-16-h1-hFc or TL-25-h1-hFc, and 0.625 μg was required for TL-34-h1-hFc to provide full protection against 10 × Lethal Dose 50 (LD<sub>50</sub>) TeNT. In the prophylactic setting, 125 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc provided full protection even when they were injected 12 days before exposure to 10 × LD<sub>50</sub> TeNT, while TL-34-h1-hFc was less effective. In the therapeutic setting, 25 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc could provide complete protection when administered 24 h after exposure to 5 × LD<sub>50</sub> TeNT, while TL-34-h1-hFc required 50 μg/kg. Conclusion: Our results suggest that TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc provide a bright future for the development of anti-TeNT preventive or therapeutic drugs.https://www.mdpi.com/2073-4468/14/2/48tetanus toxinTL-HNVHHphage-display libraryhumanized VHH-hFc fusion proteinneutralization |
| spellingShingle | Yating Li Kexuan Cheng Jiazheng Guo Yujia Jiang Qinglin Kang Rong Wang Peng Du Chen Gao Yunzhou Yu Zhixin Yang Wei Wang Jiansheng Lu Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In Vivo Antibodies tetanus toxin TL-HN VHH phage-display library humanized VHH-hFc fusion protein neutralization |
| title | Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In Vivo |
| title_full | Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In Vivo |
| title_fullStr | Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In Vivo |
| title_full_unstemmed | Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In Vivo |
| title_short | Humanized VHH-hFc Fusion Proteins Targeting the L-HN Fragment of Tetanus Toxin Provided Protection In Vivo |
| title_sort | humanized vhh hfc fusion proteins targeting the l hn fragment of tetanus toxin provided protection in vivo |
| topic | tetanus toxin TL-HN VHH phage-display library humanized VHH-hFc fusion protein neutralization |
| url | https://www.mdpi.com/2073-4468/14/2/48 |
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