Study On Candidate Melanoma Neoantigen Screening And DNA Vaccine Preparation

With the development of sequencing technology and histochemistry research,people classify tumors more accurately and formulate precision tumor treatment strategies according to the characteristics of tumor,which promotes the development of tumor neoantigen vaccine at the same time.Tumor specific neoantigens have been proved to be promising targets for personalized tumor treatment and have achieved encouraging clinical outcomes recently.Both animal tumor models and clinical trials have proved their safety and capability of inducing effective immune response against tumors.Multiple papers about accurate prediction of neoantigen have been published and many companies claim that they select neoantigens for patient using several software which will give us the best prediction result according to their knowledge.However,the positive responsive rate of neoantigen selected by these software is generally low.This is mainly due to the complex process of antigen peptide presentation in cells,which is hard for the simple linear algorithm to accurately predict.In addition,the amount of accumulated antigenic peptide fails the need of machine learning,resulting in inaccurate prediction of immunogenic neoantigen.Therefore,it is necessary to build a set of wet experiment protocol,combined with prediction result,to effectively find the potential neoantigen as vaccine candidate.DNA vaccine is superior in short preparation cycle,simple storage conditions,economic and effectiveness,which can be widely used in therapeutic tumor vaccine in the future.However,the immune response induced by naked DNA is not strong enough.Developing a reasonable carrier platform for DNA vaccine is vital to ensure the safety and improve delivery efficiency of DNA vaccine in vivo.In this study,we chose mouse melanoma cell lines B16F10 and C57BL/6 as our research objects to study the safety and effectiveness of DNA vaccine against tumor.Considering human heterogenicity in gene background,MHC polymorphism and diversity of antitumor immune response among individuals,which may have an impact on pharmacodynamic consistency evaluation,we analyzed the whole exon and transcriptome sequencing data of subcutaneous melanoma and lung metastasis melanoma obtained from C57BL/6 mice,B16F10 inoculated.We discovered that genetic background of C57BL/6mice we bought were similar among individuals,melanoma tumor tissues shared thousands of mutations,which accounted nearly 65.1% of the total mutation,but the antitumor immune response varied among individuals.Next,we selected neoantigen with algorithm prediction through setting conditions of variant allele frequency,RNA expression and higher affinity of mutated peptide with MHC molecules than wild peptide.In order to increase the amount of neoantigen peptide presented by MHC I molecules on BMDC cell surface,we added kozak-signal peptideubiquitin(KSU)sequence ahead of DNA coding neoantigen.The results of flow cytometry proved the efficiency of KSU sequence in promoting neoantigen peptide presented by MHC I molecules.Based on this gene design,10 peptides of the 54 neoantigens predicted by software were proved capable of activating stronger specific cytotoxicity of T cells towards B16F10 cells.In addition,we tried to detect the sequences of antigen peptides presented on tumor cell surface by mass spectrometry.In order to improve the anti-tumor immune response of DNA vaccine,we designed TMGIL12 co-expression DNA vaccine.Compared with TMG,TMG-IL12 could more effectively stimulate the maturation of DC cells,enhance secretions of IFN-γ and granzyme B in T cells,and induce stronger cell cytotoxicity towards B16F10 cells.Meanwhile,we prepared two kinds of liposome encapsulated DNA vaccines,DOTAP and DOTAP-DOPE,to protect DNA degradation in vivo.Gel electrophoresis results indicated that at N/P ratio 10:1,liposomes could effectively protected plasmids degradation from DNase I enzyme.Highest expression of e GFP in tumor cells was detected when tumor cells were transfected with liposome encapsulated pc DNA3.1-e GFP,which were mixed at N/P ratio 10:1 for 20 minutes at room temperature.In addition,100μg/m L liposomes showed little toxicity to BMDCs.To improve the amount of DNA vaccine uptaken by DCs in vivo,we explored the optimal concentration of Cho-Cp G ODN.The results turned out that with concentration of 0.2n M Cho-Cp G ODN(used with 5μg plasmid),the amount of liposome-DNA/Cp G nanoparticles uptaken by BMDCs within 24 hours was higher than liposome-DNA nanoparticles.At last,we detected the tumor regression efficiency of DNA vaccine in mouse melanoma models.Treatment results with different doses of DNA vaccine proved that low dose(5μg per time)could more effectively inhibit the growth of subcutaneous melanoma,promote infiltrations of CD8+ T cells and neutrophils in tumors than higher doses.There were no significant toxicities of high dose(50μg per time)of nanoparticles observed in DNA vaccine treated mouse,according to the results of body weight and immunohistochemistry of organs(liver,lung,kidney and heart).We also compared the treatment effects of D-TMG/CPG,D-TMG-IL12/CPG,DD-TMG/Cp G and DD-TMGIL12/CPG in mouse subcutaneous melanoma model and lung metastasis model,which proved that all of the four DNA vaccines could effectively inhibit subcutaneous tumor growth,prolong the life span of subcutaneous tumor bearing mice and remarkedly reduced lung metastasis.Specifically,DD-TMG-IL12/Cp G proved to be the most effective DNA vaccine in inhibiting lung metastasis.Furthermore,administration of DTMG-IL12/Cp G and DD-TMG-IL12/Cp G stimulated higher proliferation rate of CD4+T and CD8+ T cells in mouse lymph nodes than D-TMG/Cp G and DD-TMG/Cp G respectively.Here we constructed a protocol for effective selection of immunogenic tumor neoantigen.The safety and treatment efficiency of DNA vaccine were further confirmed in vitro and in vivo.Our study has established a new method for tumor neoantigen screening,provided a new idea for DNA vaccine design,and laid a foundation for the development of tumor neoantigen DNA vaccine.