Pre-clinical Study On Bio-therapy Of Malignant Tumor With Human Wild-Type P-53, GM-CSF And B7-1 Genes Mediated By Recombinant Adenovirus Vector

Malignant tumor is a kind of serious disease, it harms human being's health with it's high incidence and death rate. However, conventional therapeutic methods being used currently, such as chemotherapy, radiotherapy, surgery, all have certain limitations. It, therefore, is needed to develop a new kind of therapy for effective treatment of tumor. We had constructed a recombinant adenovirus vector mediated wild-type p53, GM-CSF and B7-1 genes (called BB-102). It can infect primary cells of laryngeal carcinoma, hepatocarcinoma, pulmonary carcinoma, mammary cancer and CML, then inhibit the tumor cells growth, and induce apoptosis and CTL. This study is focus on pre-clinical investigation of BB-102, some thing as below have been done.Part 1 Production of recombinant adenovirus, establishment of quality and quality control We draw up the discipline of production and examination of recombinant adenovirus BB-102. Produced BB-102 and Ad-GFP via 293 cell, purified with Chlorinated caesium density gradient ultracentrifugation, measured the titer by plaque assay. Test the quality according to the Chinese Biological Products Discipline and the Discipline of Production and Examination of Recombinant Adenovirus BB-102. The results indicated that the titer of Ad-GFP is 1.1 × 10¹¹ pfu/ml, BB-102 is 5 × 10¹⁰ pfu/ml, the quality of BB-102 is accord with these two disciplines.Part 2 Stability study of recombinant adenovirus BB-102 anti-tumor agent To investigate the effect of changes in temperature on storage time and protectant of recombinant adenovirus vector. Store BB-102 and Ad-GFP in Hank's solution containing 10% glycerin at different temperature (-60 ℃, — 20 ℃, room temperature,4°C and 37°C ) for different time, and store Ad-GFP in different solution such as gelatin, saccharu, lactose, glucose, maltose, sorbitol, manicol in different concentration then the viral infectivity, genes expression , transfection efficiency and the divergence of viral particle were detected. The results indicated that the quality of BB-102 and Ad-GFP stored at the temperature below — 20 °C is stable. Gelatin can be a better stable agent for store AdV at room temperature.Part 3 Effectiveness study in vivo of recombinant adenovirus BB-102 To study the treatment of a recombinant adenovirus carrying p53 gene ,B7-1 gene and GM-CSF (BB-102) on the nude mice with laryngeal carcinoma. The results showed that the difference between experimental and control groups was statistically significant. The morphology of cells infected with BB-102 was analyzed for evidence of apoptosis by transmission electron microscopy. It has been shown that wild type P53 protein can inhibit cell growth and induce apoptosis, while B7-1 and GM-CSF have no such effects. Conclusions: The results showed that recombinant adenovirus has significant efficacy on suppressing tumor cell growth and inducing their apoptosis , which suggested that BB-102 might be further developed into a therapeutic agent in clinical therapy of laryngeal carcinoma, and the suggested dose is 5 X 108 pfu / kg .BW.Part 4 Evaluation of safety of the BB-102 The result of acute toxicity test for BB-102 demonstrated that no evident toxicity reactions were found during the observation period after i.m. or i.p. injection of BB-102 to mice. The MTD of i.m. BB-102 once in mice was >4.84 X 10upfu/kg (female mice) or >4.67X 10upfu/kg (male mice ). The MTD of i.p. BB-102 once in mice was >4.87X 10upfu/kg (female mice) or >4.74X 10npfu/kg (male mice ). The result of chronic toxicity test for BB-102 demonstrated that no evident toxicity reactions were found after continuously 15 times subcutaneous injection of BB-102 with the dose of 5.0xl0810pfu/kg. Except the slightly rise on the amount of WBC, PLT and reticulocyte depended on the dose, no evident change was observed after administrated 5.0xl09pfu/kg and 5.0xl010pfu/kg via same way. It can be thought the dose below 5.0x10810pfu/kg is safe.The anti-adenovirus neutralized antibody was observed after dosed 8 times, andvanished after stop dosing 30 weeks.The recombinant adenovirus vector distributed in the skin and muscle of injectionarea, liver and lung, it had been detected AdV distributed in other organs afteradministration.The results of general pharmacological test indicated that BB-102 has no influenceon general behavior, central nervous system, cardiovascular system, respiratorysystem and vermiculation in animals.The results of allergic reaction, stimulation and hemolysis test demonstrated thatBB-102 anti-tumor agent has not system allergic reaction in Guinea pig, it did notinduce hemolysis and erythrocyte aggregation, and the stimulation to local muscle isaccord with the regulation .