Abstract
Treatment of human epidermal growth factor receptor 2 (HER2/neu)-expressing breast cancer patients with a monoclonal antibody (mAb) directed against HER2/neu improves the outcome of chemotherapy. In cases in which remission is observed, antibody-dependent cell-mediated cytotoxicity (ADCC) seems to be one of the main mechanisms of anti-HER2/neu mAb action, implicating Fc gamma receptors (Fc gamma Rs) in this tumoricidal activity. In vitro and in vivo studies have revealed that anti-HER2/neu-mediated ADCC is mainly accomplished by polymorphonuclear granulocytes (PMN). C5a, a cleavage product of the complement component C5, modulates Fc gamma R expression via upregulation of activating and downregulation of inhibitory Fc gamma Rs. C5a also recruits PMNs to sites of inflammation and increases PMN survival. To enhance the recruitment and activation of C5a receptor-bearing cells into the tumor microenvironment, we developed antibody fusion proteins composed of a human IgG3 anti-HER2/neu antibody genetically fused to C5a [anti-HER2/neu IgG3-(C5a)] or to its derivative, C5a(desArg) [anti-HER2/neu IgG3-(C5a(desArg))]. Both fusion proteins were expressed, properly assembled, and secreted by murine myeloma cells, and displayed chemotactic activity on human PMN. Under comparable conditions, anti-HER2/neu IgG3-(C5a(desArg)) increased the survival of PMN more efficiently than anti-HER2/neu IgG3-(C5a) or C5a(desArg). Surprisingly, incubation of the fusion proteins with breast cancer cells that overexpress HER2/neu (SK-BR-3) induced cell death at a dose at which the anti-HER2/neu IgG3 antibody was innocuous. In the presence of human peripheral blood leukocytes as effector cells, both fusion proteins induced tumor cell death more efficiently than anti-HER2/neu IgG3. These data suggest that anti-HER2/neu IgG3-(C5a) and anti-HER2/neu IgG3-(C5a(desArg)) fusion proteins possess novel properties that could be useful in cancer immunotherapy.
Highlights
The human epidermal growth factor receptor 2 (HER2/neu) is a 185 kDa membrane glycoprotein with intrinsic kinase activity [1]
The strategy followed for construction of the anti-HER2/ neu IgG3-(C5a) and anti-HER2/neu IgG3-(C5adesArg) expression vectors is outlined in Supplementary Fig. S1
Clones secreting anti-HER2/ neu IgG3-(C5a) or anti-HER2/neu IgG3-(C5adesArg) fusion proteins were identified by enzyme-linked immunosorbent assays (ELISA) and the proteins were examined by SDS-PAGE and Western blot
Summary
The human epidermal growth factor receptor 2 (HER2/neu) is a 185 kDa membrane glycoprotein with intrinsic kinase activity [1]. Other members of the human epidermal growth factor receptor family (HER1, HER3, and HER4) form heterodimers with HER2/neu and initiate intracellular signaling that. One experimental approach for improving the efficacy of mAb has been to fuse immunomodulatory molecules such as certain cytokines to therapeutic antibody using molecular biology techniques [5, 6]. These antibody fusion proteins seek to target immunostimulating properties of cytokine(s) into the tumor microenvironment, enhancing the positive effects of the therapy, but avoiding the adverse side effects observed after the systemic administration of some of those cytokines at high doses [7, 8]
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