Construction, Expression, And Activity Of A Genetic Engineered Anti-ErbB-2 ScFv-Fc-IL-2 Fusion Protein

Immunocytokines (ICKs) have significant potential for cancer therapy because they can be used to retarget cytokines which exhibit a wide variety of biological activities to tumor cells. In the preclinical trials using murine models, antibody-cytokine fusion proteins have shown to be very effective antitumor agents. In this study, a genetic engineered anti-ErbB-2 scFv-Fc-IL-2 fusion protein HFI was constructed. The binding characteristics and ability in vitro and in vivo killing of ErbB-2 over-expressing tumor cells of HFI were also analyzed.1 The construction of anti- ErbB-2-IL2 fusion proteins and its expression in CHO cellsBy using gene engineering technology, scFv anti-ErbB-2 cDNA, human IgG1 Fc gene, and human IL-2 gene were fused. CHO cells were transfected with the recombinant plasmid pCI/HFI. The activities of the fusion proteins in the supernatant of the transfected CHO cells were assayed by ELISA, flow cytometry and MTT methods. The engineered fusion proteins can be recognized by polyclonal antibody against human IgG and monoclonal antibody against human IL-2. The fusion proteins can bind to SKBR3 cells expressing ErbB-2 antigens, and stimulate the proliferation of IL-2 dependent CTLL2 cells. The fusion proteins retained both the ability of binding to ErbB-2 antigens and the biological activities of IL-2.2 Controlled growth of Chinese hamster ovary cells and high expression of antibody-IL-2 fusion proteins by temperature manipulationGrowth and the expression of the anti-ErbB-2 scFv-Fc-IL2 fusion protein in Chinese hamster ovary (CHO) cells were in association at 37℃. The expression of the fusion protein was no more than 25μg/ml. At 30℃the cell growth was arrested but the cells continued to produce the fusion protein up to 60-80μg/ml. About 50% of CHO cells were rapidly blocked in G2/M phase after the temperature was shifted from 37 to 30℃. Lowering temperature resulted in cell growth arrest, but maintained cell viability for a longer time and enhanced the production of the antibody-IL-2 fusion protein in CHO cells.3 Efficient Growth Inhibition of ErbB-2 Overexpressing Tumor Cells by An Anti-ErbB-2 scFv-Fc-IL-2 Fusion Protein in vitro and in vivoTumor specific antibodies genetically fused to IL-2 provide an alternative approach to improve the therapeutic index of IL-2. The underlying principle of antibody-IL-2 molecules is based on the targeting of multifunctional IL-2 to the tumor microenvironment. In the present study, a genetically engineered anti-ErbB-2 scFv-Fc-IL-2 fusion protein HFI was constructed. The fusion protein was folded as a homodimer formed by covalently linking of Fc portions, leading to a bivalent molecule, and retained ErbB-2 specificity and IL-2 biological activity. In vitro and in vivo experiments demonstrated that HFI mediated ADCC at low effector-to-target ratios and exhibited significant antitumor activity in tumor xenograft models.