Abstract LB-304: Novel antibody-(IL-12) fusion proteins with disrupted heparin-binding activity for the therapy of cancer

Abstract

Abstract Interleukin-12 (IL-12), a 70 kDa glycoprotein, is a potent immunostimulatory cytokine with a heterodimeric structure composed of two subunits, p40 and p35, that has been used for the immunotherapy of cancer. Although anti-tumor responses were observed in some patients, the systemic injection of IL-12 is associated with severe toxicity due to the high dose that is required to induce an anti-tumor immune response. In order to overcome this problem, we developed an antibody-cytokine fusion protein composed of IL-12 genetically fused to a human IgG3 specific for HER2/neu, a tumor-associated antigen that is overexpressed on breast and ovarian tumors and is associated with poor prognosis. The goal of this approach to cancer therapy is to concentrate the cytokine in the tumor microenvironment and in so doing directly enhance the tumoricidal effect of the antibody while activating a secondary anti-tumor immune response. We have shown that the antibody-(IL-12) elicits a significant anti-tumor activity in mice bearing HER2/neu expressing tumors. However, it also exhibits the heparin-binding activity of the cytokine moeity, which allows it to bind glycosaminoglycans present on the surface of a broad variety of cells and the extracellular matrix in normal tissues. This potentially reduces the capacity of the antibody-(IL-12) to specifically target HER2/neu expressing tumors due to its retention outside the tumor microenvironment, which may also result in unfavorable side effects. Therefore, the main goal of the present project is to disrupt the heparin-binding activity of the IL-12 moiety without affecting its biological activity. Since structural and functional studies have shown that the p35 subunit of IL-12 is responsible for receptor binding and signaling, we focused on the p40 subunit and predicted potential heparin-binding sites at the carboxy-terminal domain of the murine p40 subunit. This prediction was based on bioinformatic analysis of the consensus regions of the glycosaminoglycan binding site reported previously for other cytokines. Accordingly, and using site directed mutagenesis, we have developed five anti-HER2/neu antibody-cytokine fusion proteins with modifications in the potential heparin-binding sites of their IL-12 moiety. These antibody fusion proteins were expressed in murine myeloma cells and their, assembly, secretion, and binding to HER2/neu confirmed. Importantly, results obtained using flow cytometry are consistent with a decrease of heparin-binding activity in four of the mutants, which also show IL-12 bioactivity as assessed by aT-cell proliferation assay. Our results identify potential heparin-binding sites of IL-12 that are not critical for T-cell activation, and suggest that these novel antibody-cytokine fusion proteins may be effective for the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-304.