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Updated: Jun 21, 2026
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Generation of monoclonal antibody targeting fibroblast growth factor receptor 3
Olena Gorbenko1, Galyna Ovcharenko, Tetyana Klymenko
1Department of Cell Signalling, Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine.
Abstract:
Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and pathological cells and tissues.
Insights
Researchers developed antibodies targeting the Fibroblast Growth Factor Receptor 3 (FGFR3) with a specific oncogenic mutation common in bladder cancer. These antibodies can detect both mutant and wild-type FGFR3, aiding in cancer research.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Fibroblast Growth Factor Receptor 3 (FGFR3) is a receptor tyrosine kinase involved in cell growth and cancer.
- FGFR3 mutations and overexpression are linked to various cancers, including bladder cancer.
- Specific detection of mutant FGFR3 is crucial for understanding its role in tumorigenesis.
Purpose of the Study:
- To generate hybridoma clones producing monoclonal antibodies (MAbs) that specifically recognize the FGFR3/S249C mutant.
- To develop tools for distinguishing between wild-type and oncogenic FGFR3 forms.
Main Methods:
- Immunization of mice with a bacterially expressed FGFR3 extracellular domain fragment containing the S249C mutation.
- ELISA screening to identify hybridoma clones with specific antibody production.
- Western blotting and immunoprecipitation to validate antibody specificity and functionality.
Main Results:
- Several hybridoma clones initially showed specificity for FGFR3/S249C over wild-type FGFR3.
- Despite initial instability, the study successfully generated stable monoclonal antibodies recognizing both FGFR3 wild-type and FGFR3/S249C.
- The generated MAbs demonstrated specificity in Western blotting and immunoprecipitation assays using various cell systems.
Conclusions:
- The developed monoclonal antibodies are valuable tools for studying FGFR3 expression and function in both normal and cancerous cells.
- These antibodies can aid in the diagnosis and research of FGFR3-associated pathologies, particularly bladder cancer.
- Further research can utilize these antibodies to investigate the biological impact of FGFR3 mutations in cancer development.
