| 1. Construction of pHS-TRAIL expression vectorHuman tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) is a transmembrane protein consisting of281amino acids, including amino acids1-14of the intracellular domain,15-40amino acids of the transmembrane region,41-281amino acids of the extracellular domain. Since the114-281amino acids position in the extracellular domain with the pro-apoptotic activity completely, while the complete rhTRAIL is difficult to express in vitro, therefore we chose peptide114-281to construct the recombinant expression plasmid. Previous studies have found that it is difficult to decide a suitable expression system. The recombinant protein is feasible to dimerize as a non-active form when using of eukaryotic expression systems, whereas when using prokaryotic expression system, rhTRAIL protein is easy to form inclusion bodies, which make it hard to complete the post-purification and ensure product quality. Therefore, we construct the expression vector pHS-TRAIL with a tryptophan promoter and the gene of interest encoding hTRAIL114-281fragments. Then we transform the recombinant plasmid into the host bacterium W3110with the correct expression hTRAIL2. Study of the fermentation and purification processes of rhTRAILIn the previous research, we inserted the fragment that coding the extracellular114-281aa part of rhTRAILinto the downstream of tryptophan promoter on vector pHS by genetic engineering techniques. After obtaining the recombinant plasmid pHS-TRAIL, we transformed it into the host bacteria W3110. The inserted fragment was then confirmed by gene sequencing. Due to the potential large clinical dose of rhTRAIL (usually up to a hundred milligrams), the higher demands has been made with the expression system, the choice, optimization and quality control of high density fermentation process and protein purification process. Generally, the factors that affect the high density fermentation of E. coli include the supply and control of the nutrients that cell metabolism needed, the control or discharge of the toxic byproducts (lactate and ammonia), the supply of the oxygen, some key parameters, such as the real-time control of the pH value, dissolved oxygen concentration and temperature. In this chapter, we investigated and optimized the process of shake flask culture, fermentation and purification. With investigation of the composition of medium, the condition of high density fermentation, and the process of purification, we also studied to improve the amount of protein expression and the yield of the purification process, and ultimately the yield of rhTRAIL, on the promise of high quality of the biological products.3. Study of the In vitro and in vivo pharmacodynamics and mechanism of rhTRAILIn the previous study, we have explored and researched composition of medium, high-density culture optimization and purification technology, on the promise of high quality of rhTRAIL to increase the protein yield, we have obtained a sufficient amount of pure rhTRAIL. In this research, we make further research of the In vitro and in vivo pharmacodynamics and mechanism of rhTRAIL. With broad-spectrum anti-tumor activity of rhTRAIL, We used12kinds of tumor cells (HCT-116, Colo-205, A549, MCF-7an so on) to research its in vitro antitumor activity, and we have established human colon cancer HCT-116and Colo-205nude mouse models and non-small cell lung cancer95D nude mouse model, with the administration of rhTRAIL alone or with chemical drugs to research the in vivo antitumor activity of rhTRAIL. At last, we used A549cell as experimental model to study the pathway-induced apoptosis by Annexin V-propidium iodide (PI) staining and Western blot.4. Studies on the pharmacokinetics and distribution of rhTRAIL in ratsIn the previous study, we have obtained a biologically active pure rhTRAIL with genetic engineering.In this research, we injected rhTRAIL into rats at the dose of5,10and30mg/kg to study the plasma concentration time curve, injection of rhTRAIL at the dose of10mg/kg to research distribution of rhTRAIL in tissues. The results show that in the range of5～30mg/kg, the main pharmacokinetic parameters AUC and Cmax have a linear relationship with dose, and t1/2β,CL and V1do not have significant differences within the three dose groups (P>0.05). What’s more, there is no significant difference within the kinetic parameters of male and female rats. Furthermore, rhTRAIL distributed rapidly in rats after the injection. And kidney is the main organ, followed by the spleen, blood, ovary, liver and the distribution in lung, heart, brain, fat, stomach, muscle, intestine, thymus and testis tissue is low. The content of rhTRAIL in the tissues decreased rapidly with time, without any accumulation.ConclusionTRAIL is a member of TNF family, and could induce tumor cells apoptosis upon binding with different death receptors as trimer. Previous research showed that TRAIL exerted cytotoxic effect and growth inhibition towards various human tumor cell lines (e.g., lung cancer, colon cancer and so on). Although pharmaceutical companies worldwide have conducted numerous preclinical and clinical studies on TRAIL, their progresses were relatively slow along with unpronounced reasons.In this study, the recombinant TRAIL protein expression system has been constructed and a high purity of recombinant TRAIL protein has been obtained through process optimization, followed by studies on in vitro and in vivo antitumor activities, pharmacokinetics and tissue distribution. The results have demonstrated that TRAIL has good antitumor activies on various tumor cell lines and models, and kidney turns out to be the main tissue responsible for the in vivo distribution within short time (-10min). In conclusion, TRAIL has showed good drugablity according to the results above. And its short half-life problem due to fast glomerular filtration indicates the administration of TRAIL in combination or conjugation with small molecule chemical drugs may provide another promising way of TRAIL therapy. |
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