| HER2/P185 is a tyrosine kinase receptor glycoprotein, which is encoded by the her2 proto-oncogene, belongs to epidermal growth factor receptor family. Cancer is developed in normal cells and tissues when it is over expressed. It has been shown that HER2 is over-expressed in a number of human malignancies including breast, gastric, ovarian cancers, lung cancer and prostatic cancer etc. In recent years, the research of anti-HER2 antibody drugs has been a promising topic for HER2 over-expressing cancer therapy, and some monoclonal antibodies have been approved for using, such as trastuzumab and pertuzumab. Lots of results show that anti-tumor effects of anti-HER2 monoclonal antibodies are performed directly or indirectly. But in clinical, trastuzumab and pertuzumab are invalid in many cancers therapy, or cancer recurrence after a period of medical treatment. In order to meet clinical requirements and improve the therapeutic efficacy, anti-HER2 antibody drugs with novel binding epitope and mechanism are in urgent needed, as well as antibody-drug conjugates scheme. The novel engineered monoclonal antibody anti-HER-2 chimeric antibody 21(chA21), which is targeted against HER2.Our previous work alsoestablished that chA21 inhibits cell proliferation and induces apoptosis in human breast cancer SKBR3 and BT-474 cells in vitro and in vivo.HuA21 is the affinity-enhanced and humanized anti-HER2 antibody, HuA21 recognizes a conformational epitope located in subdomain I of the HER2 extracellular domainwhich derives from ChA21. HuA21 anti-tumor mechanism of in vitro and in vivo have been developed in our works through establishing mice model bearing HER2-overexpressing cancer xenografts (BT-474,SKOV3,OE-19 cell lines)and constructing HER2-overexpressing cancer cell lines with trastuzumab-resistance BT-474/HR and MCF-7/HER2,4T1-HER2-Luci cell lines. The aim is to provide experiment evidences for innovative treatment of cancer.Objective:By observing HuA21 activity against HER2 over-expressing cancer and exploring its effects on function and signal transduction of EGFR, results indicated that HuA21 led to down-regulation of EGFR receptor expression. It is to lay experimental bases for development and application of anti-HER2 humanized antibody HuA21 with novel mechanism.Methods:1. Human breast cancer cell line MCF-7 with HER2 stable transfection and BT-474/HR were constructed, respectively. And the action of HuA21 against HER2 over-expressing cancer in vitro was studied.2. Both mice models bearing MCF-7/HER2, BT-474, BT-474/HR, SKOV3 or OE-19 xenografts were established.In BT-474 and BT-474/HR group,5×106 cells (0.05 ml culture with serum and 0.05 ml Matrigel) were transplanted in the bilateral mammary fat pad of Balb/c mice, and 0.72 mg 17-beta estradiol was injected. Then they were randomly divided into PBS group, Trastuzumab group (10 mg/kg), HuA21 group (10 mg/kg) and chA21 group (10 mg/kg), with the average tumor volume of about 130-140 mm3 and BT-474/HR160 mm3, were administered twice a week at a dose of 0.1 ml.In MCF-7/HER2 group, 1×107 cells (0.1 ml culture with serum) were transplanted in the unilateral mammary fat pad of 32 female Balb/c mice, and 0.72 mg 17-beta estradiol was injected. Then 28 selected Balb/c mice were randomly divided into PBS group, Trastuzumab group (20 mg/kg), HuA21 group (20 mg/kg) and chA21 group (20 mg/kg), with the average tumor volume of about 110-130 mm3 and were administered twice a week at a dose of 0.1 ml (according to an average weight of 20g for each)In SKOV3 group,5×106 cells were transplanted in each flank of 22 female Balb/c mice. Then 20 selected Balb/c mice were randomly divided into PBS group, Trastuzumab group (30 mg/kg), HuA21 group (30 mg/kg) and chA21 group (30 mg/kg), with the average tumor volume of about 70-80 mm3 and were administered twice a week at a dose of 0.1 ml (according to an average weight of 20g for each).In OE-19 group,5×106 cells were transplanted in each flank of 26 female Balb/c mice. Then 24 selected Balb/c mice were randomly divided into PBS group, Trastuzumab group (20 mg/kg), HuA21 group (20 mg/kg) and chA21 group (20 mg/kg), with the average tumor volume of about 76-80 mm3 and were administered twice a week at a dose of 0.1 ml (according to an average weight of 20 g for each). The volumes of the xenografts were measured twice a week.The formula for tumor volume (TV):TV=1/2 ×a×b2, and the terms "a" and "b" represent length and width, respectively. The formula for relative tumor volume (RTV):RTV=Vt/V0, and V0 and Vt refer to tumor volume at initiating administration (DO) and measurement point, resp. The rate of tumor growth (T/C): T/C= (TRTV/CRTV), and TRTV and CRTV refer to the RTV of treatment group and control group, resp. All of these are as index of anti-tumor effect. The volume inhibition rate= (control -treatment)/control×100%; The weight inhibition rate= (control-treatment)/control× 100%.3. The effect of HuA21 on signaling pathways downstream of HER2 receptors in BT-474 and BT-474/HR.BT-474 and BT-474/HR cell lines were spread at 5×106 cells per dish for 48 h, respectively. Upon 10% serum, they were treated with different antibody of 10 ?g/ml (PBS, Trastuzumab, HuA21, HuA21+Trastuzumab), respectively. After treatment, cell lysates were collected for measurement of HER2, EGFR, HER3, Erk, Akt, pHER2, pHER3, pErk, pAkt and ?-acting by Western Blotting. The binding and internalization of humanized antibody HuA21 in BT-474?SKBR3 breast cancer cells and SKOV3 ovarian cancer cells were determined by immunofluorescence and confocal laser scanning microscopy, then internalization was analyzed by FACS.4. The anti-metastasis effect of HuA21 in vivo.The expression of HER2 in 4T1-HER2-Luci was analyzed by FACS, WB and in vivo imaging. And 5×105 cells were transplanted in the bilateral mammary fat pad of Balb/c mice. Then they were randomly divided into PBS group, Trastuzumab group (30 mg/kg), HuA21 group (30 mg/kg). Both administration the measurement of xenografts volumes were twice a week. Tumor metastasis was monitored by in vivo imaging system.5. Statistical AnalysiThe data were analyzed by SPSS 17.0 software. Measurement data was described with meanąsd (xąs), differences between the mean values of multiple groups were analysed by a one-way analysis of variance with Tukey's test for post hoc comparisons. Statistical significance was considered at p< 0.05.Results:1. Both cell lines MCF-7 transfected with GC-Rich/Her2 and human breast cancer cells BT-474 resistant to trastuzumab (BT-474/HR) were successfully constructed and they were used in ensuing pharmacodynamic experiments.2. HuA21 could significantly inhibited proliferation of BT-474 and BT-474/HR breast cells, SKOV3 ovarian cancer cells and OE-19 gastric cancer cells, and whose anti-proliferation activity was better than trastuzumab in BT-474/HR. Meantime the action of effector cells on tumor cells could be mediated effectively by HuA21.The researches in vivo showed that HuA21 could significantly inhibited proliferation of BT-474, BT-474/HR, MCF-7/HER2, SKOV3 and OE-19 cells,which compared with the control group.3. The researches in vitro showed that phosphorylation of Erkl/2 and Akt were down-regulated significantly by HuA21 both in BT-474 and BT-474/HR. Compared with trastuzumab, phosphorylation levels of Erkl/2, EGFR, HER2 were blocked more by HuA21 in both cells. But in BT-474/HR cells, the effect of HuA21 on down-regulating phosphorylation levels of ERK1/2 and Akt was better than trastuzumab.Humanized antibody HuA21 and Trastuzumab could specially bind to HER2 receptor on cancer cells surface, but their internalization varied greatly in different tumor cells, and HuA21 had superior efficacy.4. HuA21 has obvious inhibitory effects on proliferation and metastasis in 4T1-HER2-Luci mice.Conclusions:1. The results show that HuA21 has quite stronger inhibition on cancer cell proliferation, and certain suppression effect on HER2-overexpressing tumor which resists to trastuzumab in vivo and in vitro. Meanwhile it plays a constructive role in inhibition of proliferation and metastasis of 4T1-Luci labeled by luciferase.2. Anti-tumor mechanism of humanized antibody HuA21 may be related to down-regulation of Erk1/2 and Akt phosphorylation, and its internalization, and down-regulation of HER3, Erk1/2 and Akt phosphorylation in BT-474/HR cells.3. By exploring characteristics and mechanisms of HuA21 in inhibition of tumor proliferation and metastasis, it provide guidance for its clinical trial program. The works lay foundation for development of novel mechanism of HuA21 in tumor-fighting. |
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