Expressão, caracterização e avaliação funcional de anticorpos humanos antitetânicos

Tétano é uma doença neuromuscular causada exclusivamente pela ação da exotoxina produzida pelo Clostridium tetani (C. tetani). Apesar da existência de uma vacina segura e de baixo custo, a incidência e mortalidade do tétano ainda são preocupantes. Por isso, em casos com risco de tétano, a imunização passiva com soro contendo anticorpos antitetânicos equinos (SAT) ou com imunoglobulina humana hiperimune antitetânica (IGHAT) é utilizada. Em busca de terapia baseada na biotecnologia moderna, nosso grupo obteve anteriormente anticorpos monoclonais (mAbs) humanos antitetânicos derivados de linfócitos B isolados por single cell sorting. O objetivo deste trabalho foi ampliar e avaliar o painel de mAbs antitetânicos, visando a proposta de candidatos promissores para um futuro uso na terapia. O painel de mAbs foi testado por ELISA e Western Blot e inibição à ligação da toxina tetânica (TeNT) ao GT1b. Cinco mAbs foram analisados em microarray para identificar seus epítopos, e em ensaio de neutralização da toxina in vivo, individualmente ou associados em dois ou três. Somente uma mistura de três mAbs que se ligam a diferentes domínios da TeNT foi neutralizante, demonstrando a importância do sinergismo para a ação neutralizante. A fim de diminuir o uso de animais, alguns testes foram realizados como forma de tentar triar os anticorpos a serem testados in vivo. Porém, a execução dos testes foi limitada pela baixa concentração dos mAbs expressos e disponibilidade de TeNT, além de outras limitações dos ensaios. Devido à dificuldade em analisar um painel grande de anticorpos, decidimos nos dedicar à obtenção de uma linhagem permanente de expressão dos mAb que neutralizaram a toxina tetânica in vivo. Foi, então, encomendada a síntese de vetores já contendo sequências para a expressão das regiões constantes das cadeias pesada gama 1 e leve kappa humanas. Realizamos também a compra de vetores com as sequências otimizadas das regiões variáveis de dois mAbs para comparação com as sequências geradas no laboratório. Houve diferença entre a produtividade dos mAbs obtidos com sequências otimizadas e sequências originais. Porém, como esperado, a otimização das sequências não alterou a função dos mAbs.Tetanus is a neuromuscular disease caused exclusively by the action of the exotoxin produced by Clostridium tetani (C. tetani). Despite there is a safe and low-cost vaccine, the incidence and mortality rate of tetanus remain worrying. Therefore, in cases at risk of tetanus, passive immunization with equine anti-tetanus serum (ATS) or hyperimmune human tetanus immunoglobulin (TIG) is required. In search of therapy based on modern biotechnology, our group previously obtained human anti-tetanus monoclonal antibodies (mAbs) derived from B lymphocytes isolated by single cell sorting. The objective of this work was to expand and evaluate the panel of anti-tetanus mAbs, for the proposal of candidates for future use in therapy. The panel of mAbs was tested by ELISA, Western Blot, and tetanus toxin (TeNT) binding to ganglioside GT1b inhibition assay. Five mAbs were analyzed by TeNT peptide microarrays to identify their epitopes and by in vivo neutralization assay, individually or associated in two or three. Only when a mixture of three mAbs that bind to different domains of TeNT was neutralizing, demonstrating the importance of synergism for the neutralizing action. In order to reduce animal testing, some alternatives were performed in an attempt to screen the antibodies to be tested in vivo. However, the execution of the tests was limited by the low concentration of expressed mAbs and availability of TeNT, besides other assays limitations. Due to the difficulty in analyzing a large panel of antibodies, we decided to focus on obtaining permanent cell lines to produce the mAbs that neutralized the TeNT in vivo. We ordered the synthesis of vectors containing the sequences of the constant regions of human heavy gamma 1 and light kappa chains. We also purchased vectors with the optimized sequences of the variable regions of two mAbs in order to compare with the sequences generated in the laboratory. There was a difference between the antibodies yield obtained with the optimized sequences and original sequences. However, as expected, the optimization of the sequences did not change the function of the mAbs

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

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 FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb