Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines

Autor(es)

Manieri, Tania Maria ; Takata, Daniela Yumi ; Targino, Roselaine Campos ; Quintilio, Wagner ; Batalha-Carvalho, Joao Victor ; Lucia da Silva, Camila Maria ; Moro, Ana Maria

• Tipo de documento: Artigo Científico
• Fonte: PHARMACEUTICS; v. 14, n. 10, p. 16-pg., 2022-10-01.

Resumo

Tetanus toxin (TeNT) is produced by C. tetani, 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 C. tetani 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 Fc gamma R by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb. (AU)

• Processo FAPESP: 20/07040-1 - Centro de Pesquisa e Desenvolvimento em Imunobiológicos
• Beneficiário: Ana Maria Moro
• Modalidade de apoio: Auxílio à Pesquisa - Núcleos de Pesquisa Orientada a Problemas em São Paulo
• Processo FAPESP: 19/10724-2 - Anticorpos monoclonais recombinantes para uso terapêutico
• Beneficiário: João Victor Batalha de Carvalho
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 15/15611-0 - Anticorpos monoclonais recombinantes para uso terapêutico
• Beneficiário: Ana Maria Moro
• Modalidade de apoio: Auxílio à Pesquisa - Temático