Structural Insights into the Down-regulation of Overexpressed p185 Protein of Transformed Cells by the Antibody chA21

Abstract

p185her2/neu belongs to the ErbB receptor tyrosine kinase family, which has been associated with human breast, ovarian, and lung cancers. Targeted therapies employing ectodomain-specific p185 her2/neu monoclonal antibodies (mAbs) have demonstrated clinical efficacy for breast cancer. Our previous studies have shown that p185 her2/neu mAbs are able to disable the kinase activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185 her2/neu -overexpressing cancer cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal fragment of p185 her2/neu , which reveals that chA21 binds a region opposite to the dimerization interface, indicating that chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation of p185 her2/neu . We propose a structure-based model in which chA21 cross-links two p185 her2/neu molecules on separate homo-or heterodimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAbs to drive the receptors into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers during a "physiologic process." p185 her2/neu is one of the four receptor tyrosine kinases of the ErbB family. We initially found that both homodimerization and heterodimerization with other ErbB receptors will induce transphosphorylation of the intracellular domains and result in the downstream signaling for cell proliferation and transformation. Moreover our studies have established that heterodimerization leads to increased signaling and transforming activity (1, 2). Significant overexpression of p185 her2/neu results in abnormalities in cell signaling and can cause cell transformation. Early studies from several laboratories found that her2/neu gene was amplified and overexpressed in 20 -30% of breast and ovarian cancers. Breast cancers that have p185 her2/neu overexpressed have a more aggressive course associated with higher relapse rates (3). p185her2/neu represents the first oncoprotein target amenable for drug intervention and immunotherapy in which disabling the kinase reverses the malignant properties of the transformed cell and renders the tumor sensitive to chemotherapy and radiation therapy (4 -8).The p185 her2/neu protein possesses a similar architecture to the other three ErbB members of this family. These kinases are type 1 transmembrane proteins and comprise an extracellular domain (ECD) 4 with four subdomains (I/L1, II/S1, III/L2, IV/S2), a single transmembrane helix, an intracellular tyrosine kinase domain, and a C-terminal tail (9). Recent crystallographic studies revealed that the subdomains II and IV contribute to dimerization events of the ErbB receptors (10, 11). Monoclonal antibodies that bind the ectodomains of these ErbB proteins hav...

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