Determination Of HER2 Extracellular Segment And Related Proteins Enhancing The ADCC Function Of Herceptin

Human epidermal growth factor receptor 2(human epidermal growth factor receptor-2,HER2)is overexpressed in many solid tumors and has become a therapeutic target for HER2-positive tumors.Herceptin(Trastuzumab)is the first HER2-targeted monoclonal antibody approved by the Food and Drug Administration(FDA)for the treatment of metastatic breast cancer.Its mechanism of action is to recognize the HER2 protein and recruit nature kill(NK)cells expressing FcγRIIIa [CD16A] to attack and kill tumors by antibody-dependent cell-mediated cytotoxicity(ADCC).Objective: To determine which domain of HER2 protein is suitable to trigger ADCC function of Herceptin.Methods: Five proteins containing different domains of HER2 extracellular segment(i.e.HER2-Fc,HER2-His,T23-561-Fc,T276-T652-Fc,T276-T652-His)were eukaryotically expressed.The molecular weight and purity of the protein were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and size exclusion chromatography(SEC).Enzyme linked immunosorbent assay(ELISA)assays detect the binding of these proteins to Herceptin.Fortebio experiment verified the binding affinity between the proteins and Herceptin.NK cell activation experiments compared the ability of these five proteins to enhance the effect of Herceptin-mediated ADCC.The m RNA levels of tumor necrosis factor-α(TNF-α),granzyme and perforin were detected by q PCR.Inhibitor incorporation experiments verified whether adding inhibitors of corresponding signaling pathways could block degranulation and Interferon-γ(IFN-γ)release after NK cell activation.Results: The protein with high purity was successfully obtained.ELISA showed that HER2-Fc,HER2-His and T276-T652-His had good binding ability to Herceptin,T276-T652-Fc was poorly bound,and T23-561-Fc did not bind at all.Fortebio results showed that HER2-His and T276-T652-His had good affinity with Herceptin.NK cell activation experiments showed that HER2-Fc and T276-T652-His could significantly enhance the degranulation and IFN-γsecretion of NK cells stimulated by Herceptin in a concentration-dependent manner.q PCR demonstrated that HER2-Fc and T276-T652-His had comparable effects on NK cell activation.Inhibitor incorporation experiments showed that wortmannin,among the four inhibitors(the others were MK-2206,Rapamycin and bp V),could inhibit the degranulation and IFN-γsecretion of NK cells after activation.MK-2206 just inhibited the release of IFN-γ.Conclusion: This study demonstrates that T276-T652-His has potentials to augment ADCC function of Herceptin,which is equivalent to HER2-Fc,laying a foundation for the subsequent utilization of HER2 protein in tumor immunotherapy.