Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display
Abstract Background Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physic...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-12-01
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| Series: | Journal of Translational Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12967-023-04524-6 |
| _version_ | 1797397790313152512 |
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| author | Mahmoud Ganji Pooria Safarzadeh Kozani Fatemeh Rahbarizadeh |
| author_facet | Mahmoud Ganji Pooria Safarzadeh Kozani Fatemeh Rahbarizadeh |
| author_sort | Mahmoud Ganji |
| collection | DOAJ |
| description | Abstract Background Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physicochemical advantages over conventional mAbs rendering them suitable therapeutics and diagnostic tools. Herein, we aimed to isolate CD19-specific VHHs from a novel immune library using phage display. Methods An immune VHH gene library was constructed. Using phage display and after five biopanning rounds, two monoclonal CD19-specific VHHs were isolated. The selected VHHs were expressed, purified, and characterized in terms of their affinity, specificity, sensitivity, and ability to target CD19-positive cell lines. Moreover, in silico analyses were employed for further characterization. Results A VHH library was developed, and because the outputs of the 4th biopanning round exhibited the most favorable characteristics, a panel of random VHHs was selected from them. Ultimately, two of the most favorable VHHs were selected and DNA sequenced (designated as GR37 and GR41). Precise experiments indicated that GR37 and GR41 exhibited considerable specificity, sensitivity, and affinity (1.15 × 107 M−1 and 2.08 × 107 M−1, respectively) to CD19. Flow cytometric analyses revealed that GR37 and GR41 could bind CD19 on the surface of cell lines expressing the antigen. Moreover, in silico experiments predicted that both VHHs target epitopes that are distinct from that targeted by the CD19-specific single-chain variable fragment (scFv) FMC63. Conclusion The selected VHHs can be used as potential targeting tools for the development of CD19-based immunotherapeutics. |
| first_indexed | 2024-03-09T01:15:15Z |
| format | Article |
| id | doaj.art-078a82ba7b4c48f6ad7fe8cd5a2d9fe6 |
| institution | Directory Open Access Journal |
| issn | 1479-5876 |
| language | English |
| last_indexed | 2024-03-09T01:15:15Z |
| publishDate | 2023-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Translational Medicine |
| spelling | doaj.art-078a82ba7b4c48f6ad7fe8cd5a2d9fe62023-12-10T12:29:44ZengBMCJournal of Translational Medicine1479-58762023-12-0121112010.1186/s12967-023-04524-6Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage displayMahmoud Ganji0Pooria Safarzadeh Kozani1Fatemeh Rahbarizadeh2Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares UniversityDepartment of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares UniversityDepartment of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares UniversityAbstract Background Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physicochemical advantages over conventional mAbs rendering them suitable therapeutics and diagnostic tools. Herein, we aimed to isolate CD19-specific VHHs from a novel immune library using phage display. Methods An immune VHH gene library was constructed. Using phage display and after five biopanning rounds, two monoclonal CD19-specific VHHs were isolated. The selected VHHs were expressed, purified, and characterized in terms of their affinity, specificity, sensitivity, and ability to target CD19-positive cell lines. Moreover, in silico analyses were employed for further characterization. Results A VHH library was developed, and because the outputs of the 4th biopanning round exhibited the most favorable characteristics, a panel of random VHHs was selected from them. Ultimately, two of the most favorable VHHs were selected and DNA sequenced (designated as GR37 and GR41). Precise experiments indicated that GR37 and GR41 exhibited considerable specificity, sensitivity, and affinity (1.15 × 107 M−1 and 2.08 × 107 M−1, respectively) to CD19. Flow cytometric analyses revealed that GR37 and GR41 could bind CD19 on the surface of cell lines expressing the antigen. Moreover, in silico experiments predicted that both VHHs target epitopes that are distinct from that targeted by the CD19-specific single-chain variable fragment (scFv) FMC63. Conclusion The selected VHHs can be used as potential targeting tools for the development of CD19-based immunotherapeutics.https://doi.org/10.1186/s12967-023-04524-6CD19Cancer immunotherapyVHHNanobodymAbPhage display |
| spellingShingle | Mahmoud Ganji Pooria Safarzadeh Kozani Fatemeh Rahbarizadeh Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display Journal of Translational Medicine CD19 Cancer immunotherapy VHH Nanobody mAb Phage display |
| title | Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display |
| title_full | Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display |
| title_fullStr | Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display |
| title_full_unstemmed | Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display |
| title_short | Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display |
| title_sort | characterization of novel cd19 specific vhhs isolated from a camelid immune library by phage display |
| topic | CD19 Cancer immunotherapy VHH Nanobody mAb Phage display |
| url | https://doi.org/10.1186/s12967-023-04524-6 |
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