Expression And Puirfication Of Recombinant Target Toxin IL6_T23-PE38KDEL And Its Antitumor Effects

Tumor is the first killer of human, but we have no perfect medicine to treatmalignant tumor. Immunotoxins for targeted cancer therapy is a good strategy.Immunotoxins are protein toxins connected to an antibody or cytokine that bindsspecifically to target cells. According to the fact that IL6R has been foundoverexpression on some cancer cell surface and absent or present in very low numberon normal cells`surface, with genetic engineering techniques, we constructed gene ofrecombinant immunotoxin IL6<sub>T23</sub>-PE38KDEL by connecting gene of IL6missingN-terminal23amino acids to the gene of pseudomonas exotoxin variant (PE38KDEL).The recombinant immunotoxin IL6<sub>T23</sub>-PE38KDEL gene was successfully expressedwith biological activity in E.coli and P. pastoris. The recombinant immunotoxinIL6<sub>T23</sub>-PE38KDEL was purified and used further to evaluate its antitumor activity invitro and in vivo. The recombinant immunotoxin IL6<sub>T23</sub>-PE38KDEL exhibitsdose-dependent antitumor effects and can cause significant tumor regression withmild side effects in mice and can be produced in large scale, it may be a goodcandidate for target therapy cancer with high levels of IL6R.The construction and expression of recombinant immunotoxinIL6<sub>T23</sub>-PE38KDEL. We ligated with IL6gene which truncated N-terminal23aminoacids and gene of pseudomonas exotoxin variant (PE38KDEL) by PCR, successfullyconstructed the recombinant toxin IL6<sub>T23</sub>-PE38KDEL genes. Then, this gene wasrespectively cloned into the pET-28a and pET-22b vector to construct the E. coliexpression plasmids28a-IL6<sub>T23</sub>-PE38KDEL and22b-IL6<sub>T23</sub>-PE38KDEL. Thepositive recombinant plasmids was transformed into the E. coli BL21(DE3) strain andinduced to express IL6<sub>T23</sub>-PE38KDEL.The specific protein expressed (about56kDa)was detected by SDS-PAGE and Western blot. SDS-PAGE and Gel thin-layerscanning showed that IL6<sub>T23</sub>-PE38KDEL was expressed mainly in inclusion bodiesat37°C,amounting at approximately20%of the total protein concentration. When theinduction temperature was dropped to25°C, it was expressed in a soluble form atapproximately12.9%%of the total soluble protein concentration. Soluble expressionof recombinant toxin IL6<sub>T23</sub>-PE38KDEL have selective cytotoxicity. In improvedZYM-5052medium, recombinant strain of E.coli can product IL6<sub>T23</sub>-PE38KDELprotein by auto-induction in high-density shaking cultures. Yield of target protein istypically four-fold higher than obtained by conventional IPTG induction. We try touse Bacillus megaterium expression system to secrete recombinant toxin IL6<sub>T23</sub>-PE38KDEL. However recombinant toxin IL6<sub>T23</sub>PE38KDEL whichexpressed by recombinant strain of Bacillus megaterium in medium have not showedbiological activity to tumour cells. We also try to use pPICZaA vector and codonoptimized IL6<sub>T23</sub>-PE38KDEL gene to express recombinant IL6<sub>T23</sub>-PE38KDEL inGS115strain of P. pastoris. The activity of recombinant toxin protein was expressedand secreted in BMMY, but its expression level is too low to scale purification.Purification of IL6<sub>T23</sub>-PE38KDEL. Using inclusion body renaturation andchromatographic techniques, we set up a purification process for preparation ofrecombinant toxin IL6<sub>T23</sub>-PE38KDEL in E.coli. The recombinant immunotoxinIL6<sub>T23</sub>-PE38KDEL was expressed in E. coli cells and accumulated in inclusionbodies. The inclusion bodies were isolated and washed extensively, then refloded andpurified through some process such as Q Bio-sep HP and MonoQ chromatographyand removing endotoxin with polymyxin B, we prepared a batch of active recombinanttoxin IL6<sub>T23</sub>-PE38KDEL with high-purity and low endotoxin. Purification ofrecombinant toxin IL6<sub>T23</sub>-PE38KDEL laid a foundation for evaluation its antitumoractivity in vitro and in vivo.Specific cytotoxicity of IL6<sub>T23</sub>-PE38KDEL in vitro. We initially established aantitumor spectrum of recombinant toxin IL6<sub>T23</sub>-PE38KDEL in vitro.IL6<sub>T23</sub>-PE38KDEL can specifically kill IL6R-overexpressiong cancer cells, such asmultiple myelomas, myeloid leukemias, hepatomas and prostate carcinomas, whilethe other tests cells is not sensitive. We determined half lethal dose of sensitive cellswith MTT method. In the homologous cell lines, half-lethal dose of recombinant toxinshowed negative correlation with the number of IL6R on the cell surface. In theheterologous cell lines, it has no correlation with the number of IL6R. Binding time ofIL6<sub>T23</sub>-PE38KDEL and IL6R receptor is15<sup>3</sup>0min. IL6can competitively blockcytotoxicity of IL6<sub>T23</sub>-PE38KDEL. when concentration of IL6is twice more thanIL6<sub>T23</sub>-PE38KDEL, it`s blocking effect increased significantly.Antitumor effects of IL6_T23-PE38KDEL in vivo. We used mice abdominaltumor model to evaluate the antitumor effects of the recombinant IL6_T23-PE38KDEL.In the intravenous drug treatment experiment, the recombinant IL6_T23-PE38KDELshowed significant anti-tumor effects in vivo, can significantly prolong the survivaltime of tumor-bearing mice, complete tumor regression rate is30%. The recombinanttoxin IL6_T23-PE38KDEL exhibits dose-dependent antitumor effects in tumor-bearingmice. In the intraperitoneal drug treatment experiment, Intervention administrationshowed a significant antitumor effect, complete tumor regression rate was70%, theinhibition rate of tumor metastasis was100%. We have not found thatIL6_T23-PE38KDELwas toxic to heart, lung, spleen, kidney, intestine and brainthrough pathologic analysis except mildly elevation of transaminases and slight decline in the number of red blood cells in vivo. The results both in vitro and vivoindicated that recombinant IL6_T23-PE38KDEL may be a potential antitumor drugcandidates.