Isolation and Evaluation of Specific Human Recombinant Antibodies from a Phage Display Library against HER3 Cancer Signaling Antigen

Abstract

Background: The human epidermal growth factor receptor family comprises four homologous members: EGFR (ErbB1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4).This family plays an important role in the signaling pathway and cell proliferation. The heterodimerization of HER2 with HER3 leads to tumor cell proliferation. Monoclonal antibody to the human HER3 receptor blocks HER3 het- erodimerization and inhibits the growth of breast cancer cells. Due to their human origin, small size, rapid penetration and high affinity properties, recombinant single chain antibodies (scFv) have been introduced as the most desired agents for cancer immunotherapy. In this study, we use a phage display system to select specific scFvs against HER3 for their use in cancer targeted therapy.Methods: A phage antibody display library of scFv was panned against an immunodominant epitope of HER3. Phage rescue was performed on the library. The supernatant that contained the appropriate scFv (109 PFU/ml) was added to an immunotube which was coated with the peptide. Elution was done using log phase E. coli TG1. The clones were amplified by PCR and DNA fingerprinted to select the specific clones against the epitope. The specificity of the selected antibodies was tested in ELISA.Results: The results represented two predominant patterns with the frequency of 25%. The other patterns showed the frequencies of 5%-10%. scFv1 and scFv2 demonstrated positive ELISA with absorbances of 0.63 and 0.46, respectively while the absorbances of wells without peptide were 0.19 and 0.11, respectively.Conclusion: In this study two specific scFvs were selected against HER3 antigen in a successful panning process. Phage ELISA represented the specific binding of scFvs against HER3.The selected scFvs reacted only with the corresponding peptides. However, no reaction with the other peptides was detected. The selected anti-HER3 scFvs have suggested that these human high affinity and small antibodies that bind specifically to HER3 epitope can be considered in HER3 targeted approaches.