Antitumor Immune Effect Of Chitosan-Mediated Neoantigen Nanovaccine

Despite remarkable improvement in therapeutic and diagnosis techniques,malignant tumors remains still one of the major threats to human health.Compared to traditional treatments such as chemotherapy and radiation therapy,anti-tumor immunotherapy has the advantages to inhibit and eliminate tumor cells through precise recognization.Tumor vaccine is one of immunotherapeutic techniques,which eliminates tumor cells through induction of specific anti-tumoral immune responses.Currently,tumor vaccine,which is widely used in clinical trials,is prepared by physic mixture of adjuvants with tumor antigens.The antigen and adjuvants are easily degraded by enzymes or absorbed by plasma proteins up on vaccination,which is hard to be captured by antigen presenting cells(APCs)and move to lymph nodes for efficient immune response activation.The antigens and adjuvants can be integrated together by nanovaccine formation,which feverate APCs uptake,lymph node localization and immune response activation by preventing both antigens and adjuvants from degradation.In recent years,many nanovaccines have been developed and applied to the prevention and treatment of tumors.Tumor antigens are mainly classified into tumor associated antigens(TAA)and tumor specific antigens(TSA).Neoantigen is one type of TSAs.In comparison to TAA,TSA are exclusively expressed in cancer cells,which induces strong anti-tumor specific immuneresponses.Following the development of next generation sequencing technology and antigen affinity prediction,more and more tumor specific neoantigens were discovered and showed promising therapeutic effects in clinic.Chitosan is a natural cationic polysaccharide that is generally obtained by the deacetylation of chitin.Chitosan has excellent biosafety and immune activity.Recent reports found that chitosan can be used as adjuvant by activating the cyclic GMP-AMP synthase(c GAS)stimulator of interferon genes(STING)pathway in antigen presentation cells.Chitosan has a great application prospect as adjuvant,while it can induce both humoral and cellular immune rsponses in vivo.However,chitosan is insoluble in normal physiological states this narrowed the application scope of chitosan.We therefore constructed nanovaccine through a self-assemble approach by using neoantigen Adpgk and CpG ODN.We then screened out soluble chitosan with low molecular weight,which can activate c GAS-STING pathway.Nanovaccine was constructed by using chitosan and carboxymethyl dextran as nanomaterials by encapsulating 10K-Adpgk and CpG together.The main experimental works include the following aspects:1.Preparation of nanovaccine by using neoantigen and CpG through a self-assemble way.Mouse colon cancer neoantigen Adpgk was modified by integrating ten lysines to obtain cationic peptides.Nanovaccine was prepared by using neoantigen and CpG through a self-assemble approach,which was named PCNPs.The average size of nanovaccine is about 175nm,in which CpG ODN was protected by preventing from degradation in vitro.PCNPs could be effectively uptaken by dendritic cells(DCs)and stimulate the maturation of DCs as well as improving the efficiency of antigen cross-presentation in vitro.Efficient localization of Adpgk neoantigen into lymph nodes was observed in PCNPs vaccination mice.In addition,PCNPs vaccination induced potent cytotoxic T lymphocytes(CTL)and effector memory T cells(TEM)specific to Adpgk neoantigen in vivo.Moreover,PCNPs vaccination could offer prophylactic protection by inhibiting MC-38 tumor cell growth and significantly prolong the survival rate of mice.PCNPs could promote the infiltration of CD8~+T cells into tumor tissue with increased INF-γsecretion.2.Screening soluble chitosan in normal physiological state and evaluation of its immune effects.Soluble 1 k Da and 3 k Da chitosan was choosen for further use by detecting the solubility of chitosan with the molecular weight of 1 k Da,3 k Da,10 k Da and 100 k Da.We further evaluated the immunological activities of these two kinds of chitosan.Both 1 k Da and 3 k Da chitosan could antive BMDCs in vitro by using flow cytometry and enzyme-linked immunosorbent analysis.Experimentl data showed that both chitosan can promote the release of type I interferons,inflammatory cytokines and chemokines by activating c GAS-STING pathway,in which the related proteins were phosphorylated.Compared to 1 k Da chitosan,3 k Da chitosan is more potent in immune activation.Experimental results showed that adjuvant activity was found in chitosan by vaccination of chitosan with MC-38 cells antigens,which prevent tumor growth in mice.Chitosan promoted CTL infiltration in tumor tissues and the proliferation of CD8~+effector memory T cells.In addition,3 k Da chitosan could promote the proliferation of CD4~+T cells and promote the production of Th-1 cells dependent Ig G2c antibodies.H&E experiment showed that chitosan has good biocompatibility and is nontoxic.3.Construction of"two-adjuvant"nanocarriers encapsulating CpG and 10K-Adpgk neoantigen.CpG and neoantigen were co-encapsulated into polysaccharide nanocarriers(CANPs)formed by 3k Da chitosan and CMD.Nanocarriers with the size about 180nm could be formed by using CMD and Chi3000 with the ratio1:1.5.CpG ODN and 10K-Adpgk neoantigen can be efficiently integrated into CANPs nanocarriers by mixing CMD,Chi3000,CpG and 10K-ADPGK with the ration of 1:1:5:0.01:075.In vitro experimental data showed that CANP vaccine could prevent CpG degradation and improve phagocytosis by APCs.CANP vaccination inhibited tumor growth by inducing neoantigen specific CTL response and CD8~+memory T cells,which secreted IFN-γand TNF-αcytokines.Taken together,nanotechnology reported in this thesis has significantly improved the specific antitumor effects of neoantigen vaccines.CTL and tumor specific memory T cells were efficiently induced by nanovaccine inoculation,in which“Cold”tumors were converted into“hot”tumors by enhancing tumor infiltration of neoantigen specific CTL.However,novel approaches for tumor immunetherapy was investigated in our current studies.