Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments
Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the s...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2021-01-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2020.1864084 |
| _version_ | 1818489395514179584 |
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| author | Francesco Nannini Lenart Senicar Farhaan Parekh Khai J. Kong Alexander Kinna Reyisa Bughda James Sillibourne Xihao Hu Biao Ma Yuchen Bai Mathieu Ferrari Martin A. Pule Shimobi C. Onuoha |
| author_facet | Francesco Nannini Lenart Senicar Farhaan Parekh Khai J. Kong Alexander Kinna Reyisa Bughda James Sillibourne Xihao Hu Biao Ma Yuchen Bai Mathieu Ferrari Martin A. Pule Shimobi C. Onuoha |
| author_sort | Francesco Nannini |
| collection | DOAJ |
| description | Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics. |
| first_indexed | 2024-12-10T17:03:21Z |
| format | Article |
| id | doaj.art-0fb7f6dbbb2349a594c6d571d3da7749 |
| institution | Directory Open Access Journal |
| issn | 1942-0862 1942-0870 |
| language | English |
| last_indexed | 2024-12-10T17:03:21Z |
| publishDate | 2021-01-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj.art-0fb7f6dbbb2349a594c6d571d3da77492022-12-22T01:40:32ZengTaylor & Francis GroupmAbs1942-08621942-08702021-01-0113110.1080/19420862.2020.1864084Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragmentsFrancesco Nannini0Lenart Senicar1Farhaan Parekh2Khai J. Kong3Alexander Kinna4Reyisa Bughda5James Sillibourne6Xihao Hu7Biao Ma8Yuchen Bai9Mathieu Ferrari10Martin A. Pule11Shimobi C. Onuoha12Cancer Institute, University College London, London, UKAutolus Therapeutics, London, UKCancer Institute, University College London, London, UKCancer Institute, University College London, London, UKAutolus Therapeutics, London, UKAutolus Therapeutics, London, UKAutolus Therapeutics, London, UKGV20 Therapeutics LLC, Cambridge, MA, USAAutolus Therapeutics, London, UKAutolus Therapeutics, London, UKAutolus Therapeutics, London, UKCancer Institute, University College London, London, UKAutolus Therapeutics, London, UKPhage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics.https://www.tandfonline.com/doi/10.1080/19420862.2020.1864084Antibody discoveryrat immune librariesphage displaylong-read sequencingscFv antibodyaffinity modulation |
| spellingShingle | Francesco Nannini Lenart Senicar Farhaan Parekh Khai J. Kong Alexander Kinna Reyisa Bughda James Sillibourne Xihao Hu Biao Ma Yuchen Bai Mathieu Ferrari Martin A. Pule Shimobi C. Onuoha Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments mAbs Antibody discovery rat immune libraries phage display long-read sequencing scFv antibody affinity modulation |
| title | Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments |
| title_full | Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments |
| title_fullStr | Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments |
| title_full_unstemmed | Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments |
| title_short | Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments |
| title_sort | combining phage display with smrtbell next generation sequencing for the rapid discovery of functional scfv fragments |
| topic | Antibody discovery rat immune libraries phage display long-read sequencing scFv antibody affinity modulation |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2020.1864084 |
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