Related Experiment Video
Updated: Feb 23, 2026
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Identification of a novel single chain fragment variable antibody targeting CD24-expressing cancer cells
Shirin Eyvazi1, Bahram Kazemi2, Mojgan Bandehpour3
1Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract:
CD24 is a heavy glycosylated protein that is overexpressed in numerous cancer and cancer stem cells. It is involved in the development, invasion, and metastasis of the cancer cells. CD24 can be considered in targeted cancer therapy as a new target. Here, phage display technology was used for the identification of novel scFv antibodies against CD24. To do so, the CD24 protein was expressed and purified from a stable transgenic CHO cell line. The cells were developed by using a CD24 encoding construct, which targets the 18S rRNA gene. The recombinant CD24 was used in the biopanning process. Four rounds of biopanning were performed with the Tomlinson J library. The polyclonal phage ELISA and DNA sequencing of the selected colonies confirmed that specific binders have been enriched during the biopanning process. Based on the DNA sequencing results, three phage-displaying scFv were obtained and their specificity to CD24 was verified in the monoclonal phage ELISA. The results showed that clone 3 (called Jd3) has the highest affinity to CD24 and also is the most abundant clone (80%) among the three clones. Therefore, Jd3 was selected for further analyses. In order to produce soluble Jd3-scFv, the phage was transfected to E. coli BL21 pLysS. Soluble Jd3 scFv was expressed and purified successfully. The soluble Jd3 scFv showed appropriate affinity to recombinant CD24 in the ELISA analysis. Also, the immunocytochemistry experiment showed that the purified Jd3 scFv can bind to the CD24 expressing A549 cells. So, the scFv may be useful in the targeted delivery of drugs or diagnostic nanoparticles to the CD24 expressing cancer cells. To the best of our knowledge, this study is the first report to identify an scFv antibody against CD24, using the phage display method.
Insights
Researchers identified a novel single-chain variable fragment (scFv) antibody targeting CD24, a protein overexpressed in cancers. This CD24-specific antibody, Jd3, shows promise for targeted cancer therapy and diagnostics.
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- CD24 is a heavily glycosylated protein frequently overexpressed in various cancers and cancer stem cells.
- Its overexpression is linked to cancer development, invasion, and metastasis, making it a potential target for cancer therapy.
Purpose of the Study:
- To identify novel single-chain variable fragment (scFv) antibodies against CD24 using phage display technology.
- To evaluate the specificity and affinity of the identified scFv antibodies for potential therapeutic and diagnostic applications.
Main Methods:
- Recombinant CD24 protein was expressed and purified from a transgenic CHO cell line.
- Phage display technology, involving four rounds of biopanning with the Tomlinson J library, was employed to select CD24-specific binders.
- Selected phage clones were confirmed using polyclonal and monoclonal phage ELISA, followed by DNA sequencing.
Main Results:
- Three phage-displaying scFv antibodies against CD24 were identified, with clone 3 (Jd3) showing the highest affinity and abundance (80%).
- Soluble Jd3-scFv was successfully expressed and purified from E. coli.
- The purified Jd3-scFv demonstrated specific binding to recombinant CD24 and CD24-expressing A549 cancer cells via ELISA and immunocytochemistry.
Conclusions:
- The study reports the first identification of an scFv antibody against CD24 using phage display.
- The Jd3-scFv exhibits high affinity and specificity for CD24, suggesting its potential utility in targeted drug delivery or diagnostic nanoparticle applications for CD24-positive cancers.
