tetano
Editor, Senior Moderator
BMC Infect Dis
. 2024 Feb 13;24(1):199.
doi: 10.1186/s12879-024-09022-8. Single-domain antibodies against SARS-CoV-2 RBD from a two-stage phage screening of universal and focused synthetic libraries
Fangfang Chen[SUP] #[/SUP][SUP] 1 [/SUP], Zhihong Liu[SUP] #[/SUP][SUP] 2 [/SUP], Wei Kang[SUP] #[/SUP][SUP] 3 [/SUP], Fan Jiang[SUP] 4 [/SUP], Xixiao Yang[SUP] 1 [/SUP], Feng Yin[SUP] 2 5 [/SUP], Ziyuan Zhou[SUP] 6 [/SUP], Zigang Li[SUP] 7 8 [/SUP]
Affiliations
Background: Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobodies, as well as other single-domain antibodies (sdAbs), have been recognized as a potential diagnostic and therapeutic tool for infectious diseases. High-throughput screening techniques such as phage display have been developed as an alternative to in vivo immunization for the discovery of antibody-like target-specific binders.
Methods: We designed and constructed a highly diverse synthetic phage library sdAb-U (single-domain Antibody - Universal library ) based on a human framework. The SARS-CoV-2 receptor-binding domain (RBD) was expressed and purified. The universal library sdAb-U was panned against the RBD protein target for two rounds, followed by monoclonal phage ELISA (enzyme-linked immunosorbent assay) to identify RBD-specific binders (the first stage). High-affinity binders were sequenced and the obtained CDR1 and CDR2 sequences were combined with fully randomized CDR3 to construct a targeted (focused) phage library sdAb-RBD, for subsequent second-stage phage panning (also two rounds) and screening. Then, sequences with high single-to-background ratios in phage ELISA were selected for expression. The binding affinities of sdAbs to RBD were measured by an ELISA-based method. In addition, we conducted competition ELISA (using ACE2 ectodomain S19-D615) and SARS-CoV-2 pseudovirus neutralization assays for the high-affinity RBD-binding sdAb39.
Results: Significant enrichments were observed in both the first-stage (universal library) and the second-stage (focused library) phage panning. Five RBD-specific binders were identified in the first stage with high ELISA signal-to-background ratios. In the second stage, we observed a much higher possibility of finding RBD-specific clones in phage ELISA. Among 45 selected RBD-positive sequences, we found eight sdAbs can be well expressed, and five of them show high-affinity to RBD (EC[SUB]50[/SUB] < 100nM). We finally found that sdAb39 (EC[SUB]50[/SUB] ~ 4nM) can compete with ACE2 for binding to RBD.
Conclusion: Overall, this two-stage strategy of synthetic phage display libraries enables rapid selection of SARS-CoV-2 RBD sdAb with potential therapeutic activity, and this two-stage strategy can potentially be used for rapid discovery of sdAbs against other targets.
Keywords: Phage display; RBD; SARS-CoV-2; Single-domain antibody; Synthetic library.
. 2024 Feb 13;24(1):199.
doi: 10.1186/s12879-024-09022-8. Single-domain antibodies against SARS-CoV-2 RBD from a two-stage phage screening of universal and focused synthetic libraries
Fangfang Chen[SUP] #[/SUP][SUP] 1 [/SUP], Zhihong Liu[SUP] #[/SUP][SUP] 2 [/SUP], Wei Kang[SUP] #[/SUP][SUP] 3 [/SUP], Fan Jiang[SUP] 4 [/SUP], Xixiao Yang[SUP] 1 [/SUP], Feng Yin[SUP] 2 5 [/SUP], Ziyuan Zhou[SUP] 6 [/SUP], Zigang Li[SUP] 7 8 [/SUP]
Affiliations
- PMID: 38350843
- PMCID: PMC10865538
- DOI: 10.1186/s12879-024-09022-8
Background: Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobodies, as well as other single-domain antibodies (sdAbs), have been recognized as a potential diagnostic and therapeutic tool for infectious diseases. High-throughput screening techniques such as phage display have been developed as an alternative to in vivo immunization for the discovery of antibody-like target-specific binders.
Methods: We designed and constructed a highly diverse synthetic phage library sdAb-U (single-domain Antibody - Universal library ) based on a human framework. The SARS-CoV-2 receptor-binding domain (RBD) was expressed and purified. The universal library sdAb-U was panned against the RBD protein target for two rounds, followed by monoclonal phage ELISA (enzyme-linked immunosorbent assay) to identify RBD-specific binders (the first stage). High-affinity binders were sequenced and the obtained CDR1 and CDR2 sequences were combined with fully randomized CDR3 to construct a targeted (focused) phage library sdAb-RBD, for subsequent second-stage phage panning (also two rounds) and screening. Then, sequences with high single-to-background ratios in phage ELISA were selected for expression. The binding affinities of sdAbs to RBD were measured by an ELISA-based method. In addition, we conducted competition ELISA (using ACE2 ectodomain S19-D615) and SARS-CoV-2 pseudovirus neutralization assays for the high-affinity RBD-binding sdAb39.
Results: Significant enrichments were observed in both the first-stage (universal library) and the second-stage (focused library) phage panning. Five RBD-specific binders were identified in the first stage with high ELISA signal-to-background ratios. In the second stage, we observed a much higher possibility of finding RBD-specific clones in phage ELISA. Among 45 selected RBD-positive sequences, we found eight sdAbs can be well expressed, and five of them show high-affinity to RBD (EC[SUB]50[/SUB] < 100nM). We finally found that sdAb39 (EC[SUB]50[/SUB] ~ 4nM) can compete with ACE2 for binding to RBD.
Conclusion: Overall, this two-stage strategy of synthetic phage display libraries enables rapid selection of SARS-CoV-2 RBD sdAb with potential therapeutic activity, and this two-stage strategy can potentially be used for rapid discovery of sdAbs against other targets.
Keywords: Phage display; RBD; SARS-CoV-2; Single-domain antibody; Synthetic library.