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Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells.

ABSTRACT: Human epidermal growth factor receptor 2 (HER2) is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21) is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21) that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra), markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy.

SUBMITTER: Li R

PROVIDER: S-EPMC4849019 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells.

Li Ruilin R Hu Siyi S Chang Yan Y Zhang Zhihui Z Zha Zhao Z Huang Hui H Shen Guodong G Liu Jing J Song Lihua L Wei Wei W

International journal of molecular sciences 20160415 4

Human epidermal growth factor receptor 2 (HER2) is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21) is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21) that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibi ...[more]

PMID: 27092488

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Project description:BACKGROUND:Antibody based cancer therapies have achieved convincing success rates combining enhanced tumor specificity and reduced side effects in patients. Trastuzumab that targets the human epidermal growth factor related receptor 2 (HER2) is one of the greatest success stories in this field. For decades, trastuzumab based treatment regimens are significantly improving the prognosis of HER2-positive breast cancer patients both in the metastatic and the (neo-) adjuvant setting. Nevertheless,???50% of trastuzumab treated patients experience de-novo or acquired resistance. Therefore, an enhanced anti-HER2 targeting with improved treatment efficiency is still aspired. METHODS:Here, we determined cellular and molecular mechanisms involved in the treatment of HER2-positive BC cells with a new rabbit derived HER2 specific chimeric monoclonal antibody called "B100?. We evaluated the B100 treatment efficiency of HER2-positive BC cells with different sensitivity to trastuzumab both in vitro and in the presence of a human immune system in humanized tumor mice. RESULTS:B100 not only efficiently blocks cell proliferation but more importantly induces apoptotic tumor cell death. Detailed in vitro analyses of B100 in comparison to trastuzumab (and pertuzumab) revealed equivalent HER2 internalization and recycling capacity, similar Fc receptor signaling, but different HER2 epitope recognition with high binding and treatment efficiency. In trastuzumab resistant SK-BR-3 based humanized tumor mice the B100 treatment eliminated the primary tumor but even more importantly eradicated metastasized tumor cells in lung, liver, brain, and bone marrow. CONCLUSION:Overall, B100 demonstrated an enhanced anti-tumor activity both in vitro and in an enhanced preclinical HTM in vivo model compared to trastuzumab or pertuzumab. Thus, the use of B100 is a promising option to complement and to enhance established treatment regimens for HER2-positive (breast) cancer and to overcome trastuzumab resistance. Extended preclinical analyses using appropriate models and clinical investigations are warranted.

Project description:Autophagy has been emerging as a novel cytoprotective mechanism to increase tumor cell survival under conditions of metabolic stress and hypoxia as well as to escape chemotherapy-induced cell death. To elucidate whether autophagy might also protect cancer cells from the growth inhibitory effects of targeted therapies, we evaluated the autophagic status of preclinical breast cancer models exhibiting auto-acquired resistance to the anti-HER2 monoclonal antibody trastuzumab (Tzb). We first examined the basal autophagic levels in Tzb-naive SKBR3 cells and in two pools of Tzb-conditioned SKBR3 cells (TzbR), which optimally grow in the presence of Tzb doses as high as 200 microg/ml Tzb. Fluorescence microscopic analyses revealed that the number of punctate LC3 structures -a hallmark of autophagy- was drastically higher in Tzb-refractory cells than in Tzb-sensitive SKBR3 parental cells. Immunoblotting analyses confirmed that the lipidation product of the autophagic conversion of LC3 was accumulated to high levels in TzbR cells. High levels of the LC3 lipidated form in Tzb-refractory cells were accompanied by decreased p62/sequestosome-1 protein expression, a phenomenon characterizing the occurrence of increased autophagic flux. Moreover, increased autophagy was actively used to survive Tzb therapy as TzbR pools were exquisitely sensitive to chemical inhibitors of autophagosomal formation/function. Knockdown of LC3 expression via siRNA similarly resulted in reduced TzbR cell proliferation and supra-additively interacted with Tzb to re-sensitize TzbR cells. Sub-groups of Tzb-naive SKBR3 parental cells accumulated LC3 punctate structures and decreased p62 expression after treatment with high-dose Tzb, likely promoting their own resistance. This is the first report showing that HER2-overexpressing breast cancer cells chronically exposed to Tzb exhibit a bona fide up-regulation of the autophagic activity that efficiently works to protect breast cancer cells from the growth-inhibitory effects of Tzb. Therapeutic targeting autophagosome formation/function might represent a novel molecular avenue to reduce the emergence of Tzb resistance in HER2-dependent breast carcinomas.

Dataset Information

Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells.

Publications

Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells.

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