Maturação da afinidade de anticorpo responsivo à glicoproteína S de SARS-CoV-2 e rastreamento pela técnica de yeast surface display
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Data
2022-12-14
Autores
Nascimento, Ieda Gabriela Bernardo do
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Universidade Estadual Paulista (Unesp)
Resumo
A COVID-19 já foi confirmada em mais de 600 milhões de pessoas no mundo, acarretando mais de 6 milhões de óbitos, sendo 680 mil só no Brasil. A falta de uma farmacoterapia específica e o fato de o país ainda não ter criado um programa eficiente de testagem, evidencia a necessidade de desenvolvimento e disponibilidade de testes rápidos, de baixo custo e alta sensibilidade ao SARS-CoV-2. A proposta deste trabalho foi construir e analisar uma biblioteca de mutações aleatórias no fragmento variável da porção pesada (scFv) do anticorpo CC12.1, da linha germinativa IGHV3-53, relatado na literatura como um dos mais específicos e de maior afinidade ao domínio de ligação ao receptor (do inglês, RBD), sendo um potencial candidato para detecção específica do SARS-CoV-2. Uma biblioteca de mutantes aleatórios do anticorpo CC12.1, foi construída a partir da transformação, em linhagem de Saccharomyces cerevisiae EBY100, do inserto produzido por PCR mutagênica da sequência do anticorpo CC12.1, com o vetor pYD4 linearizado. A biblioteca de cerca de 7. 10⁶ clones foi avaliada pela técnica Yeast Surface Display (YSD) pela indução da expressão em galactose e rastreamento da interação dos fragmentos variáveis mutados com o RBD por meio de citometria de fluxo. Entre o primeiro e segundo ciclo de seleção por citometria células incubadas com 420 nM (1,05 µg/mL) de RBD, foi observado um aumento de 1,8x da fração de ligante ao RBD por fragmentos scFvs em relação a biblioteca inicial e de 13x em relação as células expressando a sequência selvagem, demonstrando potencial aplicabilidade no cenário de maturação in vitro da afinidade de anticorpos.
COVID-19 has already been diagnosed in more than 600 million people worldwide, resulting in more than 6 million deaths, 680,000 of which occurred only in Brazil. The lack of pharmacological treatment and the fact that this country has not yet created an efficient testing program highlights the need for the development and availability of quick, low-cost, high-sensitivity tests for SARS-CoV-2. The purpose of this project was to build and analyze a library of random mutations in the variable fragment of the heavy portion (scFv) of the antibody known as CC12.1, from the germline IGHV3-53, reported in the literature as one of the most specific and with the highest affinity for the Receptor Binding Domain (RBD), being a potential candidate for specific detection of SARS-CoV-2. The random mutant library of CC12.1 was constructed from the transformation of a Saccharomyces cerevisiae lineage, EBY100, from the insert produced from mutagenic PCR of the CC12.1 antibody sequence, with the linearized vector pYD4. The library, consisting of about 7. 10⁶ clones, was assessed by the Yeast Surface Display (YSD) technique, using expression induction in a medium with galactose and screening the interaction between the mutated variable fragments and the RBD using cytometry. Between the first and second cycles of selection by cytometry in cells incubated 420 nM (1,05 µg/mL) of RBD, was observed na increase of 1.8x of the binding fraction to the RBD by scFv fragments when compared to the initial library, and of 13x when taking into account the native sequence’s expression, demonstrating a potential applicability in the in vitro maturation scenario of antibody affinity.
COVID-19 has already been diagnosed in more than 600 million people worldwide, resulting in more than 6 million deaths, 680,000 of which occurred only in Brazil. The lack of pharmacological treatment and the fact that this country has not yet created an efficient testing program highlights the need for the development and availability of quick, low-cost, high-sensitivity tests for SARS-CoV-2. The purpose of this project was to build and analyze a library of random mutations in the variable fragment of the heavy portion (scFv) of the antibody known as CC12.1, from the germline IGHV3-53, reported in the literature as one of the most specific and with the highest affinity for the Receptor Binding Domain (RBD), being a potential candidate for specific detection of SARS-CoV-2. The random mutant library of CC12.1 was constructed from the transformation of a Saccharomyces cerevisiae lineage, EBY100, from the insert produced from mutagenic PCR of the CC12.1 antibody sequence, with the linearized vector pYD4. The library, consisting of about 7. 10⁶ clones, was assessed by the Yeast Surface Display (YSD) technique, using expression induction in a medium with galactose and screening the interaction between the mutated variable fragments and the RBD using cytometry. Between the first and second cycles of selection by cytometry in cells incubated 420 nM (1,05 µg/mL) of RBD, was observed na increase of 1.8x of the binding fraction to the RBD by scFv fragments when compared to the initial library, and of 13x when taking into account the native sequence’s expression, demonstrating a potential applicability in the in vitro maturation scenario of antibody affinity.
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Palavras-chave
Biblioteca de mutantes, Anticorpo scFv, SARS-CoV-2, Mutant library, scFv antibod