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Screening, expression and anti-tumor functional identification of anti-LAG-3 nanobodies
Dan Jiang1, Rui Chen1, Liyan Wang2
1Dongguan Key Laboratory of Molecular Immunology and Cell Therapy, The First Dongguan Affiliated Hospital, School of Medical Technology, Guangdong Medical University, Dongguan, Guangdong, 523808, China.
Objective:
To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor cells.
Methods:
Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjμgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc. RESULTS: One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 μg/mL.
Conclusion:
The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application.
Insights
Researchers screened and expressed a specific anti-lymphocyte activation gene-3 (LAG3) nanobody. This nanobody demonstrated an auxiliary anti-tumor cell effect by enhancing T cell-mediated tumor cell killing.
Area of Science:
- Immunology
- Biotechnology
- Oncology
Background:
- Lymphocyte-activation gene 3 (LAG3) is an immune checkpoint receptor expressed on T cells.
- LAG3 plays a role in regulating T cell responses and tumor immune evasion.
- Targeting LAG3 offers a potential strategy for cancer immunotherapy.
Purpose of the Study:
- To screen and obtain specific nanobody sequences targeting Lymphocyte-activation gene 3 (LAG3).
- To express and purify a recombinant anti-LAG3 nanobody.
- To verify the nanobody's efficacy in enhancing T cell-mediated tumor cell killing.
Main Methods:
- Utilized a camel-derived nanobody phage display library for screening.
- Employed biotin-streptavidin liquid phase screening, phage-ELISA, and sequencing to identify anti-LAG3 nanobodies.
- Expressed and purified the recombinant nanobody, followed by validation using SDS-PAGE, Western blot, and cytotoxicity assays.
Main Results:
- Successfully screened one specific anti-LAG3 nanobody sequence.
- Confirmed successful expression and purification of the recombinant anti-LAG3 nanobody.
- Demonstrated that the nanobody specifically enhances T cell-mediated tumor cell killing, with an optimal concentration of 200 μg/mL.
Conclusions:
- The developed recombinant anti-LAG3 nanobody exhibits specific and auxiliary anti-tumor activity.
- This nanobody holds promise for future applications in cancer immunotherapy.
- Provides a foundation for further research and development of LAG3-targeted therapies.
