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Production and characterization of recombinant Anti-SARS-CoV-2 Antibody Fragments (Fabs)
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O coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2) depende da interação entre o seu domínio de ligação ao recetor (RBD), na proteína Spike, e o enzima conversor de angiotensina 2 (hACE2) para a entrada celular. Desde 2019, infetou mais de 700 milhões de pessoas, causando quase 7 milhões de mortes, com variantes emergentes como a Ómicron representando uma ameaça significativa, escapando à imunidade. Apesar da importância das vacinas na mitigação da pandemia de COVID-19, estas podem não abranger todas as novas variantes, ressaltando a necessidade de antivirais e anticorpos que ataquem diretamente o vírus e reduzam a gravidade da doença. Contudo, até alguns anticorpos perdem eficácia contra novas variantes. LY-CoV555, um anticorpo descoberto no plasma de um paciente recuperado de COVID-19 e depois desenvolvido pela Eli Lilly e AbCellera, que inibe a interação entre o RBD da estirpe original do SARS-CoV-2 e o recetor hACE2, perdeu eficácia contra a subvariante Omicron BA.5. Assim, é essencial redesenhar anticorpos contra novas e dominantes variantes. Este trabalho visou produzir, purificar e caracterizar um fragmento de ligação a antigénio (Fab), computacionalmente redesenhado, derivado do LY-CoV555. O objetivo era prevenir a interação RBD-hACE2, melhorando a afinidade do Fab contra o SARS-CoV-2 Omicron BA.5. Os Fabs redesenhado e original foram produzidos com sucesso a partir de células Expi293FTM, com uma produção de 73,3 mg L-1 e 140 mg L-1, respetivamente. Ambos demonstraram estabilidade térmica (Tm = 76,1±0,2ºC para o Fab WT e 73,8±0,1ºC para o Fab Var) e perfis de agregação semelhantes (Tagg50 = 58,0±0,8ºC). Porém, a caracterização funcional por biolayer interferometry e ensaios de neutralização revelaram que o Fab redesenhado não liga nem neutraliza a Omicron BA.5. Este trabalho destacou desafios inerentes ao desenvolvimento e caracterização de fragmentos de anticorpos, abrindo caminho para investigações futuras no desenvolvimento de biofármacos contra novas variantes do SARS-CoV-2.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on the interaction between its receptor binding domain (RBD), in the Spike protein, and the human angiotensin-converting enzyme 2 (hACE2) receptor for cellular entry. Since late 2019, SARS-CoV-2 has infected over 700 million people and caused almost 7 million deaths, with emerging variants like Omicron posing a significant threat by evading immunity. While vaccines have been vital in mitigating the COVID-19 pandemic, they may not cover all emerging variants. This underlines the urgent need for antivirals and antibodies development which directly target the virus and reduce disease severity. However, even some effective antibodies developed against the original SARS-CoV-2 strain lose efficacy against new variants. LY-CoV555, an antibody discovered in the convalescent plasma of a recovered COVID-19 patient and then developed by Eli Lilly and AbCellera, that inhibit the RBD-hACE2 receptor interaction, lost its effectiveness against Omicron subvariants like BA.5. Hence, it is essential to re-design antibodies to target novel and dominant variants. This work aimed the production, purification, and characterization of a computationally re-designed fragment antigen-binding (Fab) derived from LY-CoV555. The goal was to prevent the RBD-hACE2 interaction by enhancing Fab’s binding properties to the Omicron subvariant BA.5 of SARS-CoV-2. Both the original Fab and the re-designed Fab were successfully expressed and purified from Expi293FTM cells, yielding 73.3 mg L-1 and 140 mg L-1, respectively. Both Fabs exhibited thermal stability (Tm = 76.1±0.2ºC for Fab WT and 73.8±0.1ºC for Fab Var) and similar aggregation profiles (Tagg50 = 58.0±0.8ºC). However, functional characterization by biolayer interferometry and neutralization assays revealed that the re-designed Fab didn’t have the ability to bind or neutralize the Omicron subvariant BA.5. This work tackles challenges in developing and characterizing antibody fragments, providing valuable insights that pave the way for future research in creating new biopharmaceuticals against emerging SARS-CoV-2 variants.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on the interaction between its receptor binding domain (RBD), in the Spike protein, and the human angiotensin-converting enzyme 2 (hACE2) receptor for cellular entry. Since late 2019, SARS-CoV-2 has infected over 700 million people and caused almost 7 million deaths, with emerging variants like Omicron posing a significant threat by evading immunity. While vaccines have been vital in mitigating the COVID-19 pandemic, they may not cover all emerging variants. This underlines the urgent need for antivirals and antibodies development which directly target the virus and reduce disease severity. However, even some effective antibodies developed against the original SARS-CoV-2 strain lose efficacy against new variants. LY-CoV555, an antibody discovered in the convalescent plasma of a recovered COVID-19 patient and then developed by Eli Lilly and AbCellera, that inhibit the RBD-hACE2 receptor interaction, lost its effectiveness against Omicron subvariants like BA.5. Hence, it is essential to re-design antibodies to target novel and dominant variants. This work aimed the production, purification, and characterization of a computationally re-designed fragment antigen-binding (Fab) derived from LY-CoV555. The goal was to prevent the RBD-hACE2 interaction by enhancing Fab’s binding properties to the Omicron subvariant BA.5 of SARS-CoV-2. Both the original Fab and the re-designed Fab were successfully expressed and purified from Expi293FTM cells, yielding 73.3 mg L-1 and 140 mg L-1, respectively. Both Fabs exhibited thermal stability (Tm = 76.1±0.2ºC for Fab WT and 73.8±0.1ºC for Fab Var) and similar aggregation profiles (Tagg50 = 58.0±0.8ºC). However, functional characterization by biolayer interferometry and neutralization assays revealed that the re-designed Fab didn’t have the ability to bind or neutralize the Omicron subvariant BA.5. This work tackles challenges in developing and characterizing antibody fragments, providing valuable insights that pave the way for future research in creating new biopharmaceuticals against emerging SARS-CoV-2 variants.
Descrição
Palavras-chave
SARS-CoV-2 Spike protein S-protein Receptor Binding Domain RBD Human angiotensin-converting enzyme 2