The EGF receptor (EGFR) HER3 is emerging as an attractive cancer therapeutic target due to its central position in the HER receptor signaling network. HER3 amplifies phosphoinositide 3-kinase (PI3K)-driven tumorigenesis and its upregulation in response to other anti-HER therapies has been implicated in resistance to them. Here, we report the development and characterization of RG7116, a novel anti-HER3 monoclonal antibody (mAb) designed to block HER3 activation, downregulate HER3, and mediate enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) via glycoengineering of the Fc moiety. Biochemical studies and X-ray crystallography revealed that RG7116 bound potently and selectively to domain 1 of human HER3. Heregulin binding was prevented by RG7116 at concentrations more than 1 nmol/L as was nearly complete inhibition of HER3 heterodimerization and phosphorylation, thereby preventing downstream AKT phosphorylation. In vivo RG7116 treatment inhibited xenograft tumor growth up to 90% relative to controls in a manner accompanied by downregulation of cell surface HER3. RG7116 efficacy was further enhanced in combination with anti-EGFR (RG7160) or anti-HER2 (pertuzumab) mAbs. Furthermore, the ADCC potency of RG7116 was enhanced compared with the nonglycoengineered parental antibody, both in vitro and in orthotopic tumor xenograft models, where an increased median survival was documented. ADCC degree achieved in vitro correlated with HER3 expression levels on tumor cells. In summary, the combination of strong signaling inhibition and enhanced ADCC capability rendered RG7116 a highly potent HER3-targeting agent suitable for clinical development. Cancer Res; 73(16); 5183-94. Ó2013 AACR.

No abstract

No abstract

The development of serum free medium formulation for culturing keratinocytes was a breakthrough in achieving a high number of epidermal cells for experimental and therapeutic studies, in particular to support the wound healing process. It is not clear, however, if switching the cells to highly proliferative phenotype may reflect change in other cellular functions important for the wound repair as their adhesive interactions with the extracellular matrix components. Remodelling of the extracellular matrix, particularly of fibronectin plays an essential role for guiding the cells during wound healing. The molecular mechanisms for organization of this provisional fibronectin matrix, however, are still not clear. We found that keratinocytes in serum containing medium, although in fewer numbers than fibroblasts, were able to remove adsorbed fluorescent labelled fibronectin from the substratum and reorganize it in a fibrilar pattern along the cell periphery. After 3 days the secreted fibronectin had also been organized as matrix-like fibers and as clusters deposited on the substratum after migrating cells. In contrast, serum free cultured keratinocytes fail to organize pre-adsorbed fluorescent labelled fibronectin, as well as the secreted fibronectin, although they grow very well under these conditions. Switching the cells to serum containing medium initiates the removal of fluorescent labelled fibronectin from the substratum, however without reorganization in fibrillar pattern. Most likely, these keratinocytes remove fluorescent labelled fibronectin by the expression of proteolytic activity, rather than with the mechanical function of beta(1) integrins. The latter were diffusely dispersed in serum containing conditions and tend to organize in focal adhesions in serum free cultured cells. We assumed their transient expression and different affinity state might be important for the keratinocyte migration and matrix assembly mechanism.

No abstract