Preparation And Evaluation Of Interleukin 2 Fused With Tumor Targeted Peptide As Potential Drug

Cancer immunotherapy by means of anti-tumor monoclonal antibodies, cell adoptive immunotherapy, cancer vaccine has become one of the most important strategies of tumor target therapy. Interleukin 2 (IL-2) is a cytokine with a variety of biological functions, also can enhance the B lymphoid antibody secretion and cell proliferation, which is mainly used as an immune adjuvant to mobilize the body’s immune system to control tumor in the clinic. Although the systemic administration of IL-2 was shown to stimulate antitumor responses in vivo, its efficacy has been limited by the development of serious side effects and low efficacy in the clinic. To overcome the problem, we focus on tumor targeting somatostatin analogue, octreotide, which has been researched for tumor treatment and diagnosis purpose, and engineer a novel fusion protein with the chimeric functions of somatostatin receptor targeting interleukin 2 (SIL).In this research, the fusion protein gene composed octreotide and interleukin 2 DNA sequence was successfully constructed into pET-25b vector and transformed into E.coli BL21 (DE3) to obtained the expression strain. Based on the studies of laboratoty scale, the optimized of culture medium and the scale up conditions of fermentation were developed in the bioreactors of 20L. Under the conditions of 37℃, pH 7.2, and induced with isopropy-β-D-thiogalactoside (IPTG) at the concentration of 10 mg/L when OD600 gets 20, the protein expression level beyond 30% with the final OD600 at 60.During inclusion body washing process, the cells were broken by method of enzyme lysis (0.5 %o～-l%o lysozyme,4%o deoxycholate acide sodium) combined with high pressure homogenization. In the system of 8 M urea,5 mM DTT and pH 8.6, the proteins were denatured, and the purity above 93% of SIL protein was obtained by Ni-NTA Super Flow purification and the binding capacity of Ni-NTA media for SIL protein was optimized to 10 mg/ml. The denature protein was diluted and oxidized in refolding buffer containing 4M urea, 2mM GSH-2 u M CuSO4, and the purity and yield of refolded protein were arrived to 95% and 20% respectively during refolding process.As for the tumor targeting confirmation, a serial of experiments were carried out including somatostatin receptors detection of various tumor cells, Co-immunoprecipitation experiment, immunofluorescence staining and cell binding assays in vitro. From in vitro test, it showed that SIL was still able to stimulate the proliferation of CTLL-2 cells after binding on the the tumor cells via somatostatin receptor 2 (SSTR2) that to be determined expressed on the tumor cells. In addition, the in vivo distribution of 125I-SIL in tumor burden nude mice for pharmacokinetic study showed that distribution of 125I-SIL in tumor tissue was significantly higher than other tissues, and the T/NT was greater than 1.Using the microvascular leakage (VLS) model, Evan’s blue leakage was adapted as specification to determine the degree of VLS induced by IL-2 or SIL. The experiments were carried on C57 tumor burden mice by injection of SIL and IL-2, and the results manifested that there was no significant deviation between SIL and IL-2 on inducing significant VLS. Anti-tumor study in vivo has determined the dose that IL-2 and SIL exhibit discrepancy on anti-tumor effects. Under the treating dose of 2.5×104 IU/kg, SIL exhibits significant anti-tumor effect compared with the control group and the IL-2 group (P<0.01 vs. control; P <0.05 vs. IL-2 low). Life-extension experiments on H22 tumor burden C57 mice demonstrate that 10 days survival rate after stopping treating was 100% for low dose treating group of SIL, while IL-2 treating group survival rate was only 50%.