Anti-tumor Effects Of ErbB2-targeted Engineered Antibody And Intracellular Antibody Against Breast And Ovarian Cancer And Exploration Of The Mechanisms

ErbB2 (HER2/p185her2/neu), which is encoded by the c-erbB2 (her2/neu) proto-oncogene, belongs to epidermal growth factor receptor (EGFR) family. It has been shown that ErbB2 overexpression in human malignant cancers correlates with poor prognosis and the resistance against radiotherapy, chemotherapy and endocrine therapy. Although trastuzumab/Herceptin has been widely used in patients with ErbB2-overexpressing metastatic breast cancer, many patients either do not respond to trastuzumab therapy or progress within 1 year of initiating trastuzumab treatment. Previously a tumor-inhibitory anti-ErbB2 monoclonal antibody A21, its single-chain chimeric derivative chA21 and intrabody derivative inA21 have been developed by Jing Liu Group from University of Science and Technology of China. It has been shown from the structural research that chA21 recognizes a conformational epitope in ErbB2 extracellular domain (ECD), which is distinct from that of trastuzumab and other anti-ErbB2 therapeutic antibodies, thus may represents a novel target site for anti-ErbB2 therapeutics.(1) The in vitro anti-prolifreative effects of Engineering antibody chA21 alone or its combination with paclitaxel or trastuzumab were characterized in human breast cancer cell line BT-474 and ovarian cancer cell line SKOV-3. The results suggested that enhanced growth inhibition of tumor cells occurred by chA21 in combination with paclitaxel or trastuzumab. Both mice models bearing BT-474 or SKOV-3 xenografts were established and were confirmed for their ErbB2-overexpressing by IHC method. For the dose-response relationship, the results showed that chA21 had a dose-dependent response on both BT-474 and SKOV-3 xenografts, and the most significant inhibition was found in the group given by the dose of 30 mg/kg with the average decrease of tumor-size by 62% in SKOV-3 and 69% in BT-474, as compared with their controls. So the dose of 30 mg/kg was chosen for the subsequent drug combination studies.Mice with tumor xenografts then were divided in six groups, given i.v. with normal saline, chA21 (30 mg/kg twice a week), paclitaxel (10 mg/kg once a week), trastuzumab (20 mg/kg twice a week), and a combination treatment of chA21 with paclitaxel or trastuzumab, respectively. The combination of chA21 with paclitaxel resulted in an average inhibition of SKOV-3 tumor growth by 87% and BT-474 tumor growth by 89% in comparison with their controls at day 28 (p < 0.001). However, the paclitaxel alone was 70% for SKOV-3 and 80% for BT-474 and chA21 alone was 59% for SKOV-3 and 82% for BT-474. So the combination remarkably enhanced the growth inhibition (p < 0.01). Meanwhile, the combination of chA21 and trastuzumab resulted also in a remarkable enhancement of the anti-tumor effects on both SKOV-3 and BT-474 xenografts. The combination treatment led to an average inhibition of SKOV-3 tumor growth by 89% and BT-474 tumor growth by 94% (p < 0.001), as compared with the control. This effect was significantly higher than trastuzumab alone (79% inhibition for SKOV-3 and 87% inhibition for BT-474 tumors) or chA21 alone (p < 0.01). The pathological analysis demonstrated that chA21 combined with paclitaxel or trastuzumab enhanced inhibition of the proliferation and decreased angiogenesis of tumor xenografts. Further, it is revealed that enhanced tumor growth inhibition by chA21 is associated with inhibition of ErbB2 downstream signaling such as AKT and MAPK pathways, and activation of NK cells.(2) Intrabody is one kind of antibodies that localize inside cells. Intrabody could block the specific protein which it binds, and interference the correct localization of the protein. Previously, the secretary antibody scA21 and intrabody inA21 were constructed based on the anti-ErbB2 antibody A21 scFv in our lab. In the present study, the effects of the intrabody to inhibit proliferation and down-regulate ErbB2 in ErbB2-overexpressing cancer cells were further investigated in vitro and in vivo. The results showed that the intrabody inA21 could effectively down-regulate ErbB2 expression and inhibit the proliferation of ErbB2-overexpressing cancer cells, compared with the secretary antibody scA21 or controls.In conclusion, chA21 is a new and safe tumor-inhibitory anti-ErbB2 engineering antibody. The combination treatment of chA21 with paclitaxel or trastuzumab markedly enhances the anti-tumor efficacy. This enhancement should, at least in part, be associated with the capacity of chA21 to down-regulate ErbB2 receptor, interrupt downstream signals such as MAPK and PI3K-AKT pathways, and activate natural killer cells. Therefore, chA21 may have important clinical therapeutic potential, especially for ErbB2-overexpressing cancer patients who develop resistance to chemotherapeutic agent such as paclitaxel or antibody trastuzumab. The intrabody inA21 can effectively inhibit the proliferation of ErbB2-overexpressing cancer cells in vitro and in vivo, compared with the control, and contribute to development of gene therapy for ErbB2-overexpressing cancer.