Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines
Tetanus toxin (TeNT) is produced by <i>C. tetani</i>, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status...
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| Format: | Article |
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MDPI AG
2022-09-01
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| Series: | Pharmaceutics |
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| Online Access: | https://www.mdpi.com/1999-4923/14/10/1985 |
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| author | Tania Maria Manieri Daniela Yumi Takata Roselaine Campos Targino Wagner Quintilio João Victor Batalha-Carvalho Camila Maria Lucia da Silva Ana Maria Moro |
| author_facet | Tania Maria Manieri Daniela Yumi Takata Roselaine Campos Targino Wagner Quintilio João Victor Batalha-Carvalho Camila Maria Lucia da Silva Ana Maria Moro |
| author_sort | Tania Maria Manieri |
| collection | DOAJ |
| description | Tetanus toxin (TeNT) is produced by <i>C. tetani</i>, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by <i>C. tetani</i> infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb. |
| first_indexed | 2024-03-09T19:37:05Z |
| format | Article |
| id | doaj.art-f787a8fe8b5a409089776c79aa2a332b |
| institution | Directory Open Access Journal |
| issn | 1999-4923 |
| language | English |
| last_indexed | 2024-03-09T19:37:05Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pharmaceutics |
| spelling | doaj.art-f787a8fe8b5a409089776c79aa2a332b2023-11-24T01:53:08ZengMDPI AGPharmaceutics1999-49232022-09-011410198510.3390/pharmaceutics14101985Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell LinesTania Maria Manieri0Daniela Yumi Takata1Roselaine Campos Targino2Wagner Quintilio3João Victor Batalha-Carvalho4Camila Maria Lucia da Silva5Ana Maria Moro6Biopharmaceuticals Laboratory, Butantan Institute, Sao Paulo 05503-900, BrazilBiopharmaceuticals Laboratory, Butantan Institute, Sao Paulo 05503-900, BrazilBiopharmaceuticals Laboratory, Butantan Institute, Sao Paulo 05503-900, BrazilBiopharmaceuticals Laboratory, Butantan Institute, Sao Paulo 05503-900, BrazilBiopharmaceuticals Laboratory, Butantan Institute, Sao Paulo 05503-900, BrazilBiopharmaceuticals Laboratory, Butantan Institute, Sao Paulo 05503-900, BrazilBiopharmaceuticals Laboratory, Butantan Institute, Sao Paulo 05503-900, BrazilTetanus toxin (TeNT) is produced by <i>C. tetani</i>, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by <i>C. tetani</i> infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb.https://www.mdpi.com/1999-4923/14/10/1985tetanus toxinneutralizationGT1btherapeutic antibodyvector constructionaffinity |
| spellingShingle | Tania Maria Manieri Daniela Yumi Takata Roselaine Campos Targino Wagner Quintilio João Victor Batalha-Carvalho Camila Maria Lucia da Silva Ana Maria Moro Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines Pharmaceutics tetanus toxin neutralization GT1b therapeutic antibody vector construction affinity |
| title | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
| title_full | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
| title_fullStr | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
| title_full_unstemmed | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
| title_short | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
| title_sort | characterization of neutralizing human anti tetanus monoclonal antibodies produced by stable cell lines |
| topic | tetanus toxin neutralization GT1b therapeutic antibody vector construction affinity |
| url | https://www.mdpi.com/1999-4923/14/10/1985 |
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