The Research Of Gold Nanoparticles-based High-throughput Screening Of Immunogenic Peptides

Tumor does serious harm to human health.Although scientists in the medical technology filed have been committed to cure tumor for a long time,it remains to be unconquerable till now.While the emergence of tumor immunotherapy method raises new hope for this disease.With further researches,effects of tumor immunotherapy are gradually becoming clear and benefial results have been claimed from many patients.And this treatment method has been evaluated as the top of Science's ten breakthroughs in 2013.Immunotherapy can be divided into antibody immune therapy and cellular immune therapy.In these two approaches,antibody immune therapy uses immunologic checkpoint antibody,such as PD-1 and CTLA-4,to eliminate the inhibition of on T cells from the internal environment,which enables their killing function on tumor cells;Cellular antitumor immunotherapy collects patients' own immune cells and culture them in vitro to expand and activate their immune function for transfusion back into patients.Common scenario includes DC-CTL treatment,CAR-T therapy which unitlizes chimeric antigen receptor technique,and etc.In the process of tumor immunotherapy,tumor antigens need to be correctly presented to the lymphocytes to form tumor antigen specific T cell colonies which can induce effective anti-tumor immune responses.Generally,this process is performed by antigen presenting cells such as dendritic cells(DC)to intake,process,and present antigens.Chimeric antigen receptor method embeds sequence containing tumor antigen recognition area into T cells directly through genetic modifications,thus to identify specific antigens on tumor cells and trigger immune responses.For both approaches,the key is based on correct tumor antigen(s).With the rapid progress of gene sequencing technology and development of bioinformatics and big data science,tumor heterogeneity has been continuously revealed.Therefore,how to efficiently screen immunogenic antigens from the enormous cancer mutation data is an important part of the new precision medicine concept.Computer simulation technology makes epitope prediction and high-throughput screening become practicle.Existing researches have demonstrated that the peptide sequence from simulation prediction can have stronger immunogenicity.But in practice,as limited by computing power and timing requirement,more applicable screening method is becoming necessary.Currently used methods for importing peptides into cells include electroporation transfer,liposome-mediated,and adenovirus-mediated transfer.Each method has its own disadvantages and not suitable for the purpose of high-throughput screening.Gold nanoparticles referred in this research have many clinical applications in carrying various substances into cells.Compared with other methods and biomaterials,the usage of gold nanoparticle has more advantages while its application for the screening of antigen epitope has not been reported.Therefore,this research chooses gold nanoparticles as the carrier for high-throughput screening.The screening of peptide antigen immunogenicity is achieved through its conjugation with antigen peptide,uptake by antigen presenting cell,and precisely detected by enzyme-linked immune spots(ELISPOT)assay.This research confirmed the conjugation of gold nanoparticle and synthetic peptide by spectral analysis,directly proved their entrance to cells by fluorescence and electron microscopy,verified the immunogenicity of different peptides by the secretion of IFN-? from T cells,and thus validated the feasibility of experiment design.Meanwhile,experimental parameters such as pH,buffer ingredients,and incubation time have been optimized;screening time from peptide synthesis to immunogenicity analysis has also been significantly shortened;and reaction scale has been successfully reduced to 96-well plate which realizes the ultimate goal of high-throughput screening.The platform technology of high-throughput screening of the antigen epitope involved in this project in association with computer simulations for epitope simulation will greatly promote the development and application of precision medical treatment.