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Updated: Apr 19, 2026
Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells
Published on: May 12, 2023
Generation and characterization of a bispecific diabody targeting both EPH receptor A10 and CD3
Haruhiko Kamada1, Shintaro Taki2, Kazuya Nagano3
1Laboratory of Biopharmaceutical Research, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan; The Center for Advanced Medical Engineering and Informatics, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Abstract:
The EPH receptor A10 (EphA10) is up-regulated in breast cancer but is not normally expressed in healthy tissue, thus it has been suggested that EphA10 may be a useful target for cancer therapy. This study reports a diabody, an antibody derivative binding two different target molecules, EphA10 expressed in tumor cells and CD3 expressed in T cells, which showed T cell dependent-cytotoxicity. The diabody, which has His-tagged and FLAG-tagged chains, was expressed in Escherichia coli and purified in both heterodimer (Db-1) and homodimer (Db-2) formulations by liquid chromatography. Flow cytometry analysis using EphA10-expressing cells showed that binding activity of heterodimers was stronger than that of homodimers. Addition of diabodies to PBMC cultures resulted in T-cell mediated redirected lysis, and the bioactivity was consistent with the stronger binding activity of heterodimeric diabody formulations. Our results indicate that diabodies recognizing both EphA10 and CD3 could have a range of potential applications in cancer therapy, such as breast cancers that express the EPH receptor A10, especially triple negative breast cancer.
Insights
Researchers developed a novel diabody targeting EPH receptor A10 (EphA10) on breast cancer cells and CD3 on T cells. This engineered antibody demonstrated potent T-cell mediated cancer cell killing, offering a promising new avenue for cancer therapy.
Area of Science:
- Oncology
- Immunotherapy
- Biotechnology
Background:
- EPH receptor A10 (EphA10) is upregulated in breast cancer and absent in healthy tissues, making it a potential therapeutic target.
- Targeting EphA10 could offer a selective approach for treating breast cancers, including triple-negative breast cancer.
Purpose of the Study:
- To develop and characterize a diabody that simultaneously targets EphA10 on tumor cells and CD3 on T cells.
- To evaluate the efficacy of this diabody in mediating T-cell dependent cytotoxicity against EphA10-expressing cancer cells.
Main Methods:
- Expression and purification of His-tagged and FLAG-tagged diabody in Escherichia coli.
- Characterization of diabody formulations (heterodimer Db-1 and homodimer Db-2) using liquid chromatography.
- Assessment of binding activity via flow cytometry and T-cell mediated cytotoxicity assays using peripheral blood mononuclear cells (PBMCs).
Main Results:
- Heterodimeric diabodies exhibited stronger binding affinity to EphA10-expressing cells compared to homodimeric formulations.
- Diabodies induced T-cell mediated redirected lysis of target cancer cells.
- Bioactivity correlated with the enhanced binding of heterodimeric diabodies.
Conclusions:
- Diabodies targeting both EphA10 and CD3 show significant potential for cancer immunotherapy.
- This approach is particularly relevant for breast cancers expressing EphA10, including triple-negative breast cancer.
- The engineered diabody represents a promising therapeutic strategy for redirecting T-cell activity against tumors.
