| Cancer， also called malignant tumor， can invade neighboring tissues ormetastasis to the far-end of organs to invade and destroy the body, leading to death.The main features of Carcinogenesis related to normal cell cycle regulation andapoptosis which are out of control. According to the origin of tissue, malignancyincluded epithelial carcinoma (originated in the epithelial cells), sarcoma (originatedin the connective tissue, bone and muscle, etc) and hematological malignancies.Because all kinds of cancers development fast, most cancer patients were diagnosedwith terminal cancer of which the treatments were difficult, and the results of theprognosis were not ideal, based on the clinical experience. Therefore, the cure ofcancer was a big problem in medical profession. In the modern tumor medicaltreatment, chemical medication has very important role. Most chemotherapy drugs inclinical medicine served as first-line use at present. However, these chemotherapydrugs had inhibitory or cellulotoxic effect to both tumor cells and normal cells,leading to the serious side effects in patients who usually had to use them to continuelife, which had not fundamentally improved the life quality.Along with the rapid development of the molecular biology and the geneticengineering as well as the intensive study related to molecular mechanisms oftumorigenesis, the development of the antitumor drugs has entered into a new stage,in which biotechnology antitumor drugs were appeared. They were targeted and hadmore targets, less adverse reactions and better curative effect, which made them oneof the fastest development direction of antitumor drugs. Recombinantantitumor-antivirus-protein (Trade Name: Novaferon) was a new type of consensusinterferon which did not exist in the nature but from a kind of artificial designtechnology called the DNA-shuffling and high throughput screening.Nucleotide fragments of different breakpoints and different lengths from twelveof genes of human IFN alpha subtypes, were produced by enzyme digestion. Thehomologous segments of these mixed fragments amplificated by PCR, were replaced by each other and combinated randomly to form one hundred thousand of new genes,of which a hybrid library was built.Through high throughput screening, an consensus interferon cloning strainwhose biological specific activity was much higher than that of rhIFN-α2b, wasselected from the clones expressed by new genes of the hybrid library. Due to itsadvantages of high anti-tumor activity, less side effects and the remarkable treatmenteffect to terminal malignant tumor, it had important meaning to improve both theprognosis of cancer patients and the quality of life. Therefore, quality control andantitumor mechanism research of Novaferon had great significance for it to use widelyin clinical.The first part of this research project studied the quality control of Novaferon,compared with that of rhIFN-α2b. The research of quality control includes biologicalactivity (included anti-tumor activity and anti-virus activity), purity (includedelectrophoresis assay and HPLC assay), protein content, molecular weight, isoelectricpoint, content of residual extraneous DNA, content of residual host bacterial proteins,activity of residual antibiotics, test for bacterial endotoxin, Ultraviolet spectroscopy,peptide mapping, N-terminal amino acid sequence. The study results of this partconclude as follow: the fourteen items of quality control of Novaferon were finished:according to requirments of the international standardization principles, QualityControl Points of Recombinant DNA Products and Pharmacopoeia (2010edition)Volume Ⅲ, quality control of Novaferon was studied. In addition, the method ofDaudi cell anti-proliferative assay was chosen to test the anti-tumor activity ofNovaferon. The findings indicated that:1. Quality control of Novaferon was contrasted with that of rhIFN-α2b. Purity,content of residual host bacterial proteins, activity of residual antibiotics, Ultravioletspectroscopy of the two products were the same, while anti-tumor activity, anti-virusactivity, protein content, molecular weight, isoelectric point, content of residualextraneous DNA, test for bacterial endotoxin, peptide mapping, N-terminal aminoacid sequence of them were different.2. Biological activity of Novaferon was contrasted with that of rhIFN-α2b.Obvious differences exsited in anti-tumor activity and anti-virus activity between thetwo products: anti-tumor specific activity of Novaferon was238times as many as thatof rhIFN-α2b, while anti-virus specific activity of Novaferon was10times as many asthat of rhIFN-α2b. 3. Molecular weight of Novaferon was tested, and the result was similar to thattested by MS which came from reference.4. Used in tryptic digestion of Novaferon in peptide mapping analysis, RapiGestSF, which was acid labile surfactant, resolved a problem that Novaferon can not bedigested completely by general method in Pharmacopia.The second part of this research project studied the antitumor mechanism ofNovaferon, compared with that of rhIFN-α2b. The research of antitumor mechanismincludes antitumor growth experiment in BALB/c nude, antiproliferative experimentin vitro, cell cycle arrest, apotosis, immunohistochemistry related to proliferation,apotosis and tumor angiogenesis, BIAcore analysis between the ligand (Novaferonand rhIFN-α2b) and the receptor (IFNR2). The study results of this part conclude asfollow:1. Novaferon can inhibit the tumor growth in BALB/c nude, and its inhibitoryeffect was higher than that of rhIFN-α2b.2. Novaferon can inhibit the tumor cell proliferation in vitro, and its inhibitoryeffect was higher than that of rhIFN-α2b.3. Novaferon can inhibit the tumor growth through pro-apoptotic effect andinhibition of tumor angiogenesis. Its pro-apoptotic effect and inhibition of tumorangiogenesis were higher than those of rhIFN-α2b.4. Novaferon can make S phase arrested in cell cycle of tumor cell to inhibit theproliferation and its effcet of S phase arrest in cell cycle was higher than that ofrhIFN-α2b.5. Novaferon can improve the apoptotic rate of tumor cell, and its apoptotic ratewas higher than that of rhIFN-α2b.6. The reason why the anti-tumor activity of Novaferon was higher than that ofrhIFN-α2b probably related to the higher affinity with IFNR2of Novaferon than thatof rhIFN-α2b.In conclusion, the research project related to the study of quality control andantitumor mechanism of Novaferon, compared with rhIFN-α2b.The results suggestedthat: biological specific activity of Novaferon was higher than that of rhIFN-α2b. inaddition, the antitumor mechanism of Novaferon related to S phase arrest,pro-apoptosis, inhibition of tumor angiogenesis, and these effects of it were higherthan those of rhIFN-α2b. Moreover, Novaferon had higher affinity with IFNR2thanrhIFN-α2b, which was the possible reason why Novaferon had stronger action in these antitumor mechanisms than rhIFN-α2b. The research project established thequality control methods and standards of Novaferon, and studied its mechanism ofantitumor preliminarily, whose results can provide technical support for qualitycontrol of consensus interferons and offer reference for the further development ofhigher efficacy consensus interferons. |
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