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Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display
Mahmoud Ganji1, Pooria Safarzadeh Kozani1, Fatemeh Rahbarizadeh2,3
1Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
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.
Insights
Researchers isolated two novel CD19-specific VHHs (nanobodies) using phage display. These VHHs show high affinity and specificity for CD19, offering potential for new B-cell cancer immunotherapies.
Area of Science:
- Immunology
- Biotechnology
- Oncology
Background:
- Monoclonal antibody (mAb) immunotherapies are effective for immunological and oncological conditions.
- CD19 is a key target antigen for B-cell neoplasms.
- VHHs (nanobodies) offer advantages over mAbs for therapeutic and diagnostic applications.
Purpose of the Study:
- To isolate CD19-specific VHHs from a novel immune library using phage display.
- To characterize the affinity, specificity, and targeting capabilities of isolated VHHs.
- To evaluate the potential of VHHs as CD19-based immunotherapeutic tools.
Main Methods:
- Construction and screening of an immune VHH gene library via phage display.
- Isolation and purification of CD19-specific VHHs after five biopanning rounds.
- Characterization of VHHs' affinity, specificity, sensitivity, and cell-binding ability using in vitro and in silico analyses.
Main Results:
- Two CD19-specific VHHs, GR37 and GR41, were successfully isolated and sequenced.
- GR37 and GR41 demonstrated high specificity and affinity for CD19 (1.15 × 10^7 M⁻¹ and 2.08 × 10^7 M⁻¹, respectively).
- Flow cytometry confirmed VHH binding to CD19 on cell lines; in silico analysis suggested distinct epitope targeting compared to FMC63 scFv.
Conclusions:
- The isolated VHHs, GR37 and GR41, are potent tools for targeting CD19.
- These VHHs hold promise for the development of novel CD19-based immunotherapeutics for B-cell malignancies.
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