Pharmacokinetics And Tissue Distribution Of MUC1-survivin Therapeutic Cancer Vaccine

Cancer is a serious threat to people's survival,health and quality of life due to its rapidly increasing morbidity and mortality.As the fourth tumor treatment method,tumor immunotherapy has been widely concerned because of its good therapeutic effect.With the characteristics of simple preparation,low cost and high safety,tumor gene vaccine has become the most widely studied tumor immunotherapy method.However,tumor gene vaccine as a biological agent containing antigens,which can induce the immune response of the body,generate specific immunity,and thus kill the tumor,the long-term stability and safety of it is the main research content of non-clinical testing.For the non-clinical safety testing of tumor vaccines,not only the safety content involved in toxicological perspectives such as acute toxicity tests,repeated dose toxicity tests,and allergy tests must be evaluated,also must through the organization distribution,quantitative drug absorption,distribution,metabolism and excretion in biological body,from the perspective of pharmacokinetic provide theoretical support.In this research group,a DNA vaccine(CpDV-IL2-sPD1/MS)has been constructed in the double-cistron–expression-vector,with two Tumor Associated Antigens Survivin and MUC1 gene sequences and immune adjuvant IL-2 and CpG motif.We have completed the toxicity test of single muscle injection in BALB/c mice,the whole-body active allergy test in guinea pigs,and the irritant test of intramuscular injection in rabbits,which provide relevant toxicological reference data for the clinical application of the vaccine.Based on the above-mentioned preliminary research,this paper will continue to complete the tissue distribution and pharmacokinetic research for non-clinical safety testing,so as to provide support for the clinical application of vaccines from the perspective of quantitative analysis.Firstly,we performed sequence analysis on the plasmid standard CpDV-IL2-sPD1/MS,and designed primers specifically for the sequence characteristics.Then,using the plasmid standard and mouse genomic DNA as templates,Q-PCR reactions were performed.The CT value was analyzed and the feasibility of subsequent experiments was considered.After comprehensive analysis,it was determined that the primer IL-2(Y)was selected for further experiments.After the primers are determined,the methodology is established,including optimization of the primer concentration,primer annealing temperature,and DNA load of the reaction system.During the establishment of the methodology,the single-variable method was mainly used,and the gradient test was carried out with plasmid standard as the template and sterilized water as the negative control.Finally,the reaction system of Q-PCR was determined,in which the optimal primer concentration was 200 nM,the optimal annealing temperature of the primer was 60?,and the maximum DNA load in the reaction system was 0.2 ug.Then the methodology is verified from the aspects of linearity and linear range,accuracy,precision,sensitivity,etc.Established the standard curve established by plasmid standard in the range of 10~2-10~8 copies/reaction.It has good linearity,the linear coefficient(R~2)is greater than 0.99,and the PCR amplification efficiency is between 0.9-1.1.The plasmid standard is sensitive and accurate,with a relative recovery rate between 50%-200%,an RSD of less than 20%,and a sensitivity of 62.5copies/ug genome.In summary,the establishment and verification of the overall methodology is completed.Genomic DNA of mice was extracted at different time points after administration,and Q-PCR reactions were performed using established and validated methodologies.Absolute quantitative analysis was performed on 768 experimental samples at four sampling time points in tissue samples.It can be seen from the tissue distribution results that after the administration of the vaccine,the vaccine was first detected at the injection site and in the blood,and then distributed in the selected 16 tissue sites,and there was still a little residual in the brain tissue.From this,it can be judged that after the vaccine injection,the metabolism in the body is characterized by rapid absorption and distribution and slow elimination.In summary,the research in this thesis fills in the blanks of tissue distribution and pharmacokinetics research in the non-clinical safety test of the DNA vaccine CpDV-IL2-sPD1/MS developed by the research group.For the first time,Q-PCR method was used The absolute quantitative analysis of the vaccine content further confirmed the safety of the vaccine,and also provided the main directions for the next step of the research.It provided a scientific and reliable theoretical basis for the vaccine to enter the clinical trial stage.