RESUMO
The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has posed a significant global health concern due to its severe respiratory illness and high fatality rate. Currently, despite the potential for resurgence, there are no specific treatments for MERS-CoV, and only supportive care is available. Our study aimed to address this therapeutic gap by developing a potent neutralizing bispecific antibody (bsAb) against MERS-CoV. Initially, we isolated four human monoclonal antibodies (mAbs) that specifically target the MERS-CoV receptor-binding domain (RBD) using phage display technology and an established human antibody library. Among these four selected mAbs, our intensive in vitro functional analyses showed that the MERS-CoV RBD-specific mAb K111.3 exhibited the most potent neutralizing activity against MERS-CoV pseudoviral infection and the molecular interaction between MERS-CoV RBD and human dipeptidyl peptidase 4. Consequently, we engineered a novel bsAb, K207.C, by utilizing K111.3 as the IgG base and fusing it with the single-chain variable fragment of its non-competing pair, K111.1. This engineered bsAb showed significantly enhanced neutralization potential against MERS-CoV compared to its parental mAb. These findings suggest that K207.C may serve as a potential candidate for effective MERS-CoV neutralization, further highlighting the promise of the bsAb dual-targeting approach in MERS-CoV neutralization.
Assuntos
Anticorpos Biespecíficos , Anticorpos Neutralizantes , Anticorpos Antivirais , Coronavírus da Síndrome Respiratória do Oriente Médio , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Humanos , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Animais , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Monoclonais/imunologia , Ligação Proteica , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/imunologia , Camundongos , Testes de NeutralizaçãoRESUMO
Immunoglobulin G-based monoclonal antibodies (mAbs) have been effective in treating various diseases, but their large molecular size can limit their penetration of tissue and efficacy in multifactorial diseases, necessitating the exploration of alternative forms. In this study, we constructed a phage display library comprising single-domain antibodies (sdAbs; or "VHHs"), known for their small size and remarkable stability, using a total of 1.6 × 109 lymphocytes collected from 20 different alpacas, resulting in approximately 7.16 × 1010 colonies. To assess the quality of the constructed library, next-generation sequencing-based high-throughput profiling was performed, analyzing approximately 5.65 × 106 full-length VHH sequences, revealing 92% uniqueness and confirming the library's diverse composition. Systematic characterization of the library revealed multiple sdAbs with high affinity for three therapeutically relevant antigens. In conclusion, our alpaca sdAb phage display library provides a versatile resource for diagnostics and therapeutics. Furthermore, the library's vast natural VHH antibody repertoire offers insights for generating humanized synthetic sdAb libraries, further advancing sdAb-based therapeutics.
Assuntos
Camelídeos Americanos , Biblioteca de Peptídeos , Anticorpos de Domínio Único , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Animais , Camelídeos Americanos/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Ensaios de Triagem em Larga Escala/métodos , Afinidade de Anticorpos , Técnicas de Visualização da Superfície Celular/métodosRESUMO
The rapid emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in the ongoing global coronavirus disease 2019 (COVID-19) pandemic. Thus, the rapid development of a platform to detect a broad range of SARS-CoV-2 variants is essential for successful COVID-19 management. In this study, four SARS-CoV-2 spike protein-specific single-chain variable fragments (scFvs) were isolated from a synthetic antibody library using phage display technology. Following the conversion of these scFvs into monoclonal antibodies (mAbs) (K104.1-K104.4) and production and purification of the mAbs, the antibody pair (K104.1 and K104.2) that exhibited the highest binding affinity (K104.1 and K104.2, 1.3 nM and 1.9 nM) was selected. Biochemical analyses revealed that this antibody pair specifically bound to different sites on the S2 subunit of the spike protein. Furthermore, we developed a highly sensitive sandwich immunoassay using this antibody pair that accurately and quantitatively detected the spike proteins of wild-type SARS-CoV-2 and multiple variants, including Alpha, Beta, Gamma, Delta, Kappa, and Omicron, in the picomolar range. Conclusively, the novel phage display-derived mAbs we have developed may be useful for the rapid and efficient detection of the fast-evolving SARS-CoV-2.