Design,Synthesis And Immune Evaluation Of MUC1 Glycopeptide Tumor Vaccine And Its Delivery System

Synthetic tumor vaccines are prepared by synthesizing tumor-related antigen fragments with clear structures by chemical,biological and other methods,and then covalently combining them with different immune stimulators.Synthetic vaccines have clear structures,simple preparation,easy regulation,safety and non-toxicity,and generally do not require additional immune adjuvants.Therefore,the development of synthetic tumor vaccines has become an important direction of tumor therapy.Mucin type I（MUC1）is overexpressed on the surface of various tumor cells,such as breast cancer,pancreatic cancer,prostate cancer,etc.,and it appears abnormal glycosylation.MUC1 is a potent tumor-associated antigen and is therefore considered an ideal target for tumor vaccine development.As a tumor marker,tumor-associated MUC1antigen is being developed as a tumor vaccine for clinical trials.However,MUC1 antigen is an autoantigen with low immunogenicity.Moreover,the metabolic stability of tumor associated carbohydrate antigen on its surface is poor,which is easy to be hydrolyzed by glycosidase in vivo,resulting in the reduction of the recognition of antigen by the immune system,so it is unable to produce a strong specific immune response.These problems have restricted the development of MUC1 tumor vaccine.The immunogenicity of the vaccine can be improved in varying degrees by combining MUC1 antigen with carrier protein,connecting T helper cell epitope peptide,binding different immune stimulants or unnatural modification of natural antigen.However,the MUC1 vaccine that can really break the antigen immune tolerance and applied to tumor treatment has not been developed.In response to this scientific question,this paper selected the most immunogenic domain in MUC1 glycopeptide（PDTRP）as the research object,and the glycosylation site threonine（Thr）was modified with tumor associated carbohydrate antigen Tn or 6F-Tn antigen.It was then covalently linked toα-Galactosylceramide（α-GalCer）,the ligand of i NKT cells to construct a well-defined two-component self-adjuvant fully synthetic tumor vaccine.Finally,the vaccine was prepared into nano-liposomes for delivery,and the immune activity of the vaccine was evaluated.The vaccine is designed by replacing natural antigens with fluorine atoms,introducing self-adjuvantα-GalCer,and using nanoliposome delivery system to enhance the immunogenicity of antigen and improve the efficiency of antigen presentation,thereby stimulating a strong immune response.The main research content of this paper includes the following four aspects:Firstly,the synthesis of tumor associated carbohydrate antigens Tn,TF and their fluorinated derivatives.Tn-Thr block was synthesized by chemical method.The 6F-Tn-Thr block was synthesized by introducing fluorine atom efficiently by microwave assisted technology.Gal K and Bi Gal Hex NAc P,two highly expressed enzymes for TF antigen synthesis,were obtained by E.coli.The synthesis system of TF-Thr block by"one pot and two enzymes"method was constructed.TF-Thr and 6F-TF-Thr antigen blocks were synthesized with high yields of 84%and 76%,respectively.Secondly,the synthesis ofα-GalCer and its amino derivative 6NH₂-α-GalCer.A variety of reaction routes for the synthesis ofα-GalCer were designed.By optimizing the reaction route and screening the reaction conditions,the reaction efficiency was improved,and higher yield ofαconfiguration products were obtained.In order to covalently bind the adjuvantα-GalCer to the MUC1 antigen,the 6NH₂-α-GalCer block was designed by introducing amino group into the 6-position of the galactose ofα-GalCer,and the block was efficiently synthesized by orthogonal protective group strategy.Thirdly,preparation of MUC1 glycopeptide tumor vaccine and its liposome delivery system.The PDT（α-Tn）RP glycopeptide antigen was synthesized by the method of solid-phase peptide synthesis,and the two-component vaccine candidate was obtained after covalently linking it with the vaccine adjuvant 6NH₂-α-GalCer.Based on the amphiphilic structure of glycopeptide lipid vaccines,nano liposome vaccine was prepared by thin film dispersion method.The pharmaceutical evaluation showed that the particle sizes of natural MCU1 and fluorinated MUC1 liposome vaccines were 108.3 nm and 105.0 nm,respectively,with uniform particle size distribution.And the introduction of fluorine atom greatly increases the electronegativity of liposome surface.Fourthly,the immune activity evaluation of MUC1 glycopeptide tumor vaccine.Balb/c mice were immunized in this paper.The antibody titer,antibody typing and cytokine secretion in the serum of immunized mice were determined by ELISA.Among them,fluorinated liposome vaccine showed the highest Ig G antibody titer level（22400）,and the main types of Ig G antibodies were Ig G1,Ig G2a,Ig G2b,etc.Comparing the ratio of Ig G2a and Ig G1 in each vaccine group,it was found that fluorinated liposome vaccine was more likely to promote Th1 immune response.The secretion of cytokines IFN-γand IL-4 in serum demonstrated that the vaccine mediated both cellular and humoral immunity.The binding ability of antibody serum to tumor cell MCF-7 was determined by flow cytometry.It was proved that fluorinated vaccine could produce cross recognition and strong binding to natural MUC1 antigen.The cytotoxicity of antibody serum was determined by MTT assay.It was proved that the antibody serum induced apoptosis of tumor cells by activating the complement system to produce cytotoxicity.In conclusion,a self-adjuvant fully synthetic tumor vaccine based on MUC1glycopeptide antigen was designed and synthesized in this paper,and delivered to mice in the form of nano liposomes for immunization.Preliminary immunological results showed that fluorinated MUC1 nanoliposome vaccine had good immunological effect.It is expected to be a promising tumor vaccine worthy of further study.In addition,through the derivation of natural antigen,the introduction of endogenous adjuvants,the use of nano-liposome delivery system,the physical and chemical properties of vaccines can be changed from different aspects,enhance the immunogenicity of vaccines,and then improve the immune response ability of the body.These research strategies provide reference for the development of synthetic tumor vaccine.