Background: Monoclonal antibodies (mAbs) are clinically used to treat several types of cancer due to their high specificity and potential to activate immune systems. The therapeutic effects of mAbs are unsatisfactory, however, in cases of aggressive cancer or low expression level of target protein. Recently, various antibody formats has been developed through genetic engineering technologies to improve therapeutic effect. Most notably bispecific antibodies (BsAb) are a highly promising form of target therapy. A BsAb that simultaneously targets the antigens on cancer cells and activated T-cells reportedly has the potential to show cytotoxicity more effectively than conventional mAbs. In our efforts to develop a new bispecific monoclonal antibody drug against breast cancer, we attempted to generate a tandem single-chain variable fragment (taFv) targeting both the T-cell CD3 antigen and Ephrin receptor A10 (EphA10), which were previously identified as breast cancer novel biomarker proteins.
Methods: We constructed the taFv (EphA10/CD3) expression vector that contain two single chain Fv (scFv) derived from the anti-EphA10 antibody and anti-CD3 antibody joined by a flexible five amino acid linker (G4S). The taFv was expressed transiently in CHO-S cells and verified for identity by Western blot analysis, was purified via its C-terminal His tag by metal ion affinity chromatography (IMAC). Flow cytometry was used to confirm dual specificity of taFv against cells expressing the appropriate targets. The ability of cytotoxic for taFv from PBMC was analyzed by lactate dehydrogenase (LDH) release assay.
Results: The taFv monomer was purified from CHO-S cells together with the taFv dimer. By flow cytometry analysis, each monomer and dimer bound both antigens EphA10 and CD3 while binding to CD3 was lower than full-body IgG. And each taFvs showed highly cytotoxicity specifically against EphA10 over-expression cancer cell line. Most notably, the cytotoxicity of taFv dimer was higher than taFv monomer at low Effector/Target ratio and low concentration.
Conclusion: In this research, it's proved that this taFv (EphA10/CD3) could induce EphA10 specific cytotoxicity. So, we are now ongoing to evaluate the therapeutic effects of each taFvs (monomer and dimer) in vivo.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C239.
Citation Format: Shintaro Taki. Development of EphA10/CD3 bispecific tandem scFv as a drug candidate against breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C239.