Antitumor Effect Of A New Anti-HER2 Monoclonal Antibody And Its Mechanism Of Action

Human epidermal growth factor receptor-2(HER2,or ErbB2),a member of ErbB family,plays key roles in several signaling pathways of breast cancer.Studies have revealed that HER2 is overexpressed in 25%-30% of human breast cancer,and is closely associated with tumor invasion,progression,and the higher recurrent rate in patients.Signaling occurs through ligand-dependent activation by external ligands like epidermal growth factor(EGF)and transforming growth factor ?(TGF?)or ligand-independent activation by receptor dimerization through interaction with other HER family members(EGFR?HER3?HER4).Through intrinsic intracellular protein tyrosine kinase activity,ErbB receptor dimerizations cause autophosphorylation within the cytoplasmic domain and thereby downstream signaling via pathways like the mitogen-activated protein kinase(MAPK)and phosphatidylinositol 3-kinase(PI3K)/Akt pathways.In recent years,HER2 targeted therapy has become a hot topic in the treatment of breast cancer.Trastuzumab(Herceptin,Genentech,San Francisco,CA,USA)is a recombinant humanized monoclonal immunoglobulin G1 kappa antibody against the extracellular domain of HER2 protein.This anti-HER2 antibody was approved by the Food and Drug Administration(FDA)on September 25,1998 as the first HER2-targeted therapy drug for metastatic breast cancer patients whose tumors overexpressed the HER2 protein.The use of trastuzumab has led to significant improvements in survival in ErbB2-positive breast cancer.However,over 70% patients still do not respond to trastuzumab treatment.Pertuzumab(Omnitarg)is anther recombinant,humanized monoclonal antibody that acts as a HER2 dimerization inhibitor by binding to the extracellular dimerization domain II.Heterodimerization with other HER2 family members like HER3 is blocked.In contrast,trastuzumab had little effect on ligand-mediated ErbB2/ErbB3 association.The results of two combined trastuzumab adjuvant therapy trials showed significantly improved clinical outcomes among the patients with surgically removed HER2 positive breast cancer.In 2012,FDA has approved Trastuzumab plus pertuzumab as treatment for patients.Despite the effectiveness of combination therapy,the objective response rate is only 24.2%,and less than 8% of patients experience a complete response.Thus,there is still an urgent need to develop new ErbB2-directed antibodies.In our study,the extracellular domain of HER2(HER2-ECD)was constructed,expressed and purified.Then,HER2-ECD protein was used to immunize female Balb/c mice.Finally,a mouse anti-HER2 monoclonal antibody,7C3,was obtained by the hybridoma technique.The competitive binding assay showed that both Trastuzumab and Pertuzumab did not compete with 7C3 for binding to HER2 suggesting that 7C3 recognizes a distinct epitope from Trastuzumab and Pertuzumab.The co-immunoprecipitation experiment was performed to evaluate the ability of 7C3 to inhibit the formation of HER2 heterodimer.The results showed that 7C3 was able to effectively inhibit ErbB2–EGFR complex formation in ErbB2-overexpressing breast cancer cell line SK-BR-3 and BT-474.We next examined the effect of 7C3 on HER3 signaling pathways by by Western blot analysisand found that 7C3 significantly decreased the level of p-MAPK but have no significant effect on the phosphorylation of Akt.Furthermore,we evaluated the ability of 7C3 to inhibit the in vitro proliferation of BT-474 and SK-BR-3 breast cancer cell lines.Our results indicated that 7C3 was able to effectively suppress breast cancer cell proliferation.To further identify the epitope recognized by 7C3,the 7C3 Fab was prepared and crystalized with HER2-ECD protein.The crystal structure of the HER2 in complex with 7C3 Fab was refined to 3.1 ? resolution.The crystal structure data showed that 7C3 Fab directly bound to the domains II/III of HER2,away from the dimerization arm..Interestingly,in our crystal structure,we observed a conformation change in 7C3 bound HER2 compared with Free HER2.We speculate that 7C3 could block the formation of HER2 heterodimerization and downstream signaling pathways by inducing HER2 conformational change by binding to domain II / III,although 7C3 did not directly bind the dimerized arm of HER2.In summary,we define a new class of anti-HER2 monoclonal antibody.,Unlike all other anti-HER2 antibodies,it can induce a major conformational change of HER2.Although it does not directly bind to HER2 dimerization arm,it can effectively inhibit dimerization of HER2,block the HER2 signaling pathways,and then inhibit the growth of HER2-overexpressing breast cancer cells.This study further elucidates the relationship between the structure and function of HER2,and provides a new HER2 targeting antibody agent for the treatment of breast cancer.This new anti-HER2 antiboy may have the potential to be combined with other HER2 targeting antibodies to improve the efficacy of HER2-targeting theray.