Screening Of The Recombinant Toxin Targeted CCK2R Efficiently And Analysis Of Its Anti-tumor Activity And Applicable Gastric Cancer Case

Gastric cancer is the most common gastrointestinal cancer and one of the leadingcauses of cancer-related death in humans. The mortality rate of gastric cancer ranksecond only to lung cancer and liver cancer in China. It is reported there is an annualincrease of up to several million cases of gastric cancer, China accounts for about42%; the annual number of global gastric cancer-related deaths is about800,000,China accounts for35%. The conventional treatments for gastric cancer includesurgery, chemotherapy, radiotherapy, immune therapy and so on. Surgical resection isthe best choice to cure gastric cancer, however not all patients with gastric cancer aresuitable for surgery, and it is very easy to relapse and metastase after the operation,prognosis is poor; chemotherapy and radiotherapy play important roles in the adjuvanttreatments of gastric cancer, but they have sevious side effects, which will destroypatient’s immune system easily; immune therapy such as cancer vaccines, most ofwhich are still under development or in clinical trials, their effectiveness are unknown.In recent years, targeted molecular therapy has become a new hot spot in the field ofcancer therapy, as it is consistent with the characteristic of gastric cancer such asobvious heterogeneity, complex biological behavior and so on. Targeted moleculartherapy can block signal transduction pathway through specifically targeting oradjusting the molecules expressed aberrantly on tumor cells, then inhibiting thegrowth, invasion or metastasis of tumor cells, it shows advantages of strongspecificity and high selectivity comparing to the traditional treatments such aschemotherapy. The main purpose of this study was to construct and screen arecombinant toxin that targeted cholecystokinin type II receptor (CCK2R), obtainedthe druggability evaluation of this recombinant toxin including anti-tumor activity,toxicology, safety and so on by cytotoxicity assay and animal studies, and determinedits clinical value, finally established a diagnostic method for its applicable clinicalcases, we hoped to lay a foundation for future clinical trials and provide guidance for designing a personalized treatment plan for gastric cancer.In our previous studies, a gastric cancer-targeted immunotoxins rG17PE38hadbeen successfully constructed based on the affinity changes and significant expressiondifference of CCK2R between gastric cancer and normal cells. In order to screen arecombinant toxin with smaller molecular weight and better activity, we tookN-terminal truncated gastrin G17(G13and G14) and N-terminal extendedoctapeptide cholecystokinin CCK8(CCK9and CCK12) as oriented units respectively,and fused with a modified form of Pseudomonas extoxin (PE38) to construct8recombinant toxins. These8kinds of recombinant toxins were all epressed by E. coliin soluble form, nevertheless the cytotoxicity experiment showed that only4recombinant toxins constructed with the reversed rG13, rG14, rCCK9and rCCK12could kill several gastric cancer cell lines and colon cancer cells lines, and both theexpression levels and cytoactivities of them were inferior to that of rG17PE38,therefore rG17PE38was chosen to perform the subsequent experiments.Firstly, rG17PE38was purified by saturated ammonium sulfate precipitation soas to remove most of the impurities, followed by hydrophobic chromatography andion exchange chromatography, finally desalted by gel filtration chromatography. Theconditions for ammonium sulfate precipitation (such as saturation of ammoniumsulfate, pH value, precipitation time, etc.) and chromatography (such aschromatographic medium, pH, salt concentration, etc.) were optimized. See from theresults, the purity of rG17PE38could reach50%after precipitation by40%saturatedammonium sulfate; and92%after hydrophobic and ion exchange chromatography;the final purity reached95%or so after desalting, meeting the requirements ofsubsequent experiments.For convenient preservation, the purified rG17PE38was lyophilized, and thelyophilization conditions were also optimized. CCK-8toxicity testing kit was utilizedto analyze the activity and stability of the lyophilized protein; an indirectimmunofluorescence method was taken to determine the position and internalztionprocesses of rG17PE38on target cells; the binding of rG17PE38with its receptor wasanalyzed by cytotoxicity assay; at last, whether rG17PE38could induce apoptosis wasverified by flow cytometry. The results showed the activity of lyophilizated rG17PE38did not decrease much, and maintained good stability at4℃for at least10days;rG17PE38could target cell surface quickly by identifying CCK2R, then internalized into cells and killed the target cells; one of the mechanisms for rG17PE38showedcytoactivity was inducing apoptosis.To further analyze the anti-tumor activity of rG17PE38, a gastric cancer nude mousemodel was established and treated by rG17PE38to observe its antitumor effects,influence on mice survival, side effects and obtain safety evaluation. The resultsdemonstrated the gastric cancer mouse model was successfully established15days afterinoculation of BGC-823cells subcutaneously, the treatment was divided into high,medium and low dose groups, among which high-dose and medium-dose groups couldinhibit tumor growth and metastasis and the survival time of mice was significantlyprolonged; moreover the pathological results of sliced tissues showed that rG17PE38hadno significant side effects on nude mice. It could be predicted rG17PE38was a promisingtargeted drug for gastric cancer based on the above results.Finally, in order to limit the scope of clinical cases targeted by rG17PE38, so thatwe will be able to treat gastric cancer patients accurately in the future, we preliminallyestablished a method to detect CCK2R. RT-PCR, western-blotting and real-time PCRmethods were used respectively to detect CCK2R in a variety of cells and gastriccancer samples qualitatively and quantitatively, we analyzed the relationship betweenCCK2R expression level and rG17PE38validity, and predicted the clinical casesapplicabe to rG17PE38. There were a total of5kinds of cancer cells and5cases ofgastric cancer samples detected the existence of CCK2R in20kinds of cells and20clinical gastric cancer samples, the results detected by PCR and western-blotting wereexactly the same. According to cytotoxicity experiments, animal studies and clinicalpathological diagnosis of gastric cancer patients, we provided a preliminary forecastthat number14,1and20patients may be suitable for rG17PE38therapy.To conclude, we confirmed the ability of rG17PE38to specifically target andresist gastric cancer from cell and animal levels respectively through the experimentsmentioned above, hoping the results in our experiments could provide reference forfuture clinical trials and designing an individualized treatment program for gastriccancer.