Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Huang, Ganggang; Zhong, Zhenwei; Miersch, Shane; Sidhu, Sachdev S.; Hou, Shin-Chen; Wu, Donghui

doi:10.3791/57357

Cited by 5 publications

(6 citation statements)

References 28 publications

(11 reference statements)

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“…It was observed that only a portion of the library's clones have all CDRs randomized. All this leads to no more than 50% efficiency of the mutagenesis (11).…”

Section: Discussionmentioning

confidence: 99%

“…Kunkel mutagenesis and electrocompetent cells preparation were performed according to the Sidhu lab protocol (11). Purified CCC-dsDNA was used in RCA for template elimination and mutated DNA amplification (12).…”

Section: Synthetic Vhh Library Generationmentioning

confidence: 99%

“…For the affinity maturation of the G12 clone a FR1-CDR1-FR-2-CDR2 region was randomized by subcloning this region from pooled synthetic libraries phagemids into the G12 sequence. The resulted diversity was electroporated into E.coli ER2738 pre-infected with M13KO7 phage for G12-based library construction as described elsewhere (11). Three rounds of biopanning were performed with different RBD concentration from 10 nM to 100 pM as described above for SARS-CoV-2 binders identification.…”

Section: Vhh Affinity Maturation and Tandem Repeats Formationmentioning

confidence: 99%

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Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library

et al. 2022

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The COVID−19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.

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“…It was observed that only a portion of the library's clones have all CDRs randomized. All this leads to no more than 50% efficiency of the mutagenesis (11).…”

Section: Discussionmentioning

confidence: 99%

Section: Synthetic Vhh Library Generationmentioning

confidence: 99%

Section: Vhh Affinity Maturation and Tandem Repeats Formationmentioning

confidence: 99%

See 1 more Smart Citation

Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library

et al. 2022

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“…The resulted diversity was electroporated into E . coli ER2738 pre-infected with M13KO7 phage for G12-based library construction as described elsewhere (28). Three rounds of biopanning were performed with different RBD concentration from 10 nM to 100 pM as described above for SARS-CoV-2 binders identification.…”

Section: Methodsmentioning

confidence: 99%

“…For the affinity maturation of the G12 clone FR1-CDR1-FR-2-CDR2 region was randomised by subcloning this region from pooled synthetic libraries phagemids into the G12 sequence. The resulted diversity was electroporated into E.coli ER2738 pre-infected with M13KO7 phage for G12-based library construction as described elsewhere (28). Three rounds of biopanning were performed with different RBD concentration from 10 nM to 100 pM as described above for SARS-CoV-2 binders identification.…”

Section: Vhh Affinity Maturation and Tandem Repeats Formationmentioning

confidence: 99%

Development of an Escape-resistant SARS CoV-2 Neutralizing Synthetic Nanobody

Dormeshkin

Shapira

Dubovik

et al. 2022

Preprint

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An emerging COVID-19 pandemic resulted in a global crisis, but also accelerated vaccine development and antibody discovery. In this work, we identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD), by screening synthetic humanized antibody library with more than 1e11 diversity. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, their affinities are weakly influenced by SARS-CoV-2 VoC mutations. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies respectively for emerging Omicron variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.

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