• Antitumor evaluation of mutant type of interleukin 2 in comparison to wild type IL-2
  • Rada Dehghan,1,* Arezoo Beig Parikhani,2 Yeganeh Talebkhan,3 Mohammad Ali Shokrgozar,4 Reza Ahangari Cohan,5 Mahdi Behdani,6
    1. Venom and Biotherapeutics Molecules Laboratory, Department of Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
    2. Venom and Biotherapeutics Molecules Laboratory, Department of Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
    3. Department of Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
    4. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
    5. Department of Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran.
    6. Venom and Biotherapeutics Molecules Laboratory, Department of Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.


  • Introduction: Interleukin-2 (IL-2), a member of the γ-chain cytokines with a molecular weight of 15kDa compromising of 133 amino acids and four alpha-helices, has been considered as an important cytokine in the survival, expansion, and function of CD8+ regulatory T cells, and natural killer cells in immunotherapy and treatment of melanoma and renal cell carcinomas. IL-2 is generally produced by CD4+ cells to activate CD8+ T and NK cells. It plays a crucial role in the induction of both cellular and humoral immunity through proliferation and activation of antigen-activated T cells, stimulation of NK cells, and homeostasis. Aldesleukin (Proleukin®) is known as one of the first FDA-approved cytokines in immunotherapy of metastatic renal cell carcinoma and malignant melanoma. However low efficacy of IL-2 therapy in cancer patients is due to the short serum half-life which causes administration of higher doses of IL-2 (720,000 IU/kg) and consequently, severe toxicity and vascular leak syndrome will occur. In addition, the interaction of IL-2with its alpha receptor and expansion of regulatory T (Treg) cells will activate immunosuppressive responses and reduces T cell-mediated anti-tumor activities.
  • Methods: mutated human IL-2 with a reduced affinity towards high-affinity IL-2Rα (CD25) was designed by selective amino acid substitutions. The amino acid sequence of the wild IL-2 and mutant IL-2 protein were separately subcloned into the pET28a expression vector. BL21 (DE3) E. coli strain was transformed with recombinant vectors. The expression of wild and mutant hIL-2 proteins was induced with 0.5mM isopropyl β-D-1-thiogalactoside (IPTG) at OD600nm and incubated for 6 hours. After purification on Ni–NTA agarose column (ABT) at a flow rate of 1ml/min. Protein concentration was determined by UV adsorption at 280nm using a spectrophotometer. The purity of the eluted protein was analyzed on 12% sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue staining. For western blotting, proteins were transferred to a nitrocellulose membrane. The membrane was treated with HRP-conjugated anti-His antibody and the protein bands were visualized using 3,3 diaminobenzidine tetrahydrochloride (DAB) solution as the substrate. We investigated the antitumor efficacy and cytotoxicity of the designed human IL-2 mutant rather than wild-type IL-2 (wtIL-2). Furthermore, we investigated the antitumor efficacy and cytotoxicity of the designed human IL-2 mutant rather than wtIL-2. Fifteen female C57BL/6J mice (6-8 weeks, 20g) were purchased from the animal resource center (Pasteur Institute of Iran) and maintained under standard housing conditions. TC1 cell line (mouse tumor cell line) cultured in DMEM medium supplemented with 10% FBS at 37°C under 5% CO2 atmosphere. On day 0, 1×106 cells in PBS (pH7.4) were injected subcutaneously (s.c) into the right flank of mice, and tumor growth was daily examined till the tumor dimension reached to 50 mm. After scarification of the animals, solid tumor samples were thinly sliced (2 mm thick) and subcutaneously transplanted into the shaved right flank of 15 mice which were divided into three groups (5 mice per group). Mutant and wild-type groups received recombinant purified IL-2 proteins which were diluted in sterile PBS (pH7.4) to a final concentration of 1 mg/kg was injected at the site of the tumor, two times per week for 1 month. The control group received 200µl PBS. Tumor size in each group was measured using a caliper and tumor volume was calculated.
  • Results: To evaluate the antitumor activity of the expressed IL-2 proteins, tumor-bearing C57BL/6 mice were treated with 1mg/kg of wild and mutant forms of IL-2 protein. The tumor size was measured for approximately 1 month after tumor transplantation and tumor volume was calculated. It was observed that tumor growth in animals treated with wild or mutant IL-2 proteins was significantly inhibited compared to the PBS treated control mice. Observations revealed that tumor volume of the mutant IL-2-treated mice was significantly decreased in comparison with the wild IL-2 treated group.
  • Conclusion: In accordance with the previous studies, it was found that reducing the interaction of IL-2 with its alpha receptor increases in vivo antitumor activity of this cytokine. Therefore, it seems that mutant IL-2 can be a promising agent for the treatment of cancer since it does not show the main side effects of IL-2-based immunotherapy.
  • Keywords: Interleukin-2, antitumor, regulatory T cells.