Preparation Of Injectable Hydrogel Tumor Vaccine And Study Of Its Effect On Tumor Immunotherapy

Immunotherapy as a specific treatment method has shown good prospects in the field of tumor treatment.So far,although immune cell therapy has achieved a good therapeutic effect on specific hematological tumors,current solid cell tumor therapy has only shown some potential for eliminating tumor cells and does not completely cure cancer.In recent years,biomaterial scaffolds for sustained release of immune cell stimulators have shown good effects in in situ recruitment and regulation of antigen-presenting cells and lymphocytes.The core goal of this study is to use a biocompatible hydrogel as antigen,immunomodulator delivery vehicle,or immune cell culture matrix,in combination with tumor antibody therapy or immune cell therapy,and to regulate the tumor microenvironment.Through this combined immunotherapy strategy,a tumor vaccine formulation with clinical conversion value is obtained.In recent years,as a new type of tumor vaccine,antigenic epitopes have received great attention in the field of personalized immunotherapy of tumors.However,the epitope peptide has a small molecular weight and a simple chemical structure,resulting in a weak immunogenicity,which is easily degraded in the body,and can not induce a sufficient intensity of immune response,and the anti-tumor effect is not obvious.Through chemical modification and self-assembly techniques,a novel epitope vaccine was designed and constructed to improve epitope immunogenicity and specific cellular immune responses,thereby promoting the development of personalized cancer vaccines and tumor immunotherapy.Aims:In the aspect of biological material carrier,a hydrogel carrier with flexible properties can be obtained by regulating the chemical composition of the polymer material and applied to tumor immunotherapy.In tumor immunotherapy,through the combination of therapeutic tumor vaccines and immune checkpoint inhibitors,it is expected to promote autoimmune responses of the body,reduce tumor cell immunosuppression,and use double-antigen epitope hydrogel vaccine to further improve cancer immunotherapy.As a result,vaccine with clinical conversion potential will be obtained.Methods:First of all,the mPEG-b-poly(L-proline)(PEV)diblock polymer was prepared by ring-opening polymerization(ROP)of L-valine N-carboxylic anhydride(L-val NCA).By varying the degree of polymerization of L-Val-NCA,the relationship between length of polypeptide chains and secondary structure,self-assembly behavior and hydrogel properties was investigated.The mouse fibroblast NIH/3T3 cells were then used to examine the cytotoxicity of a series of hydrogels and the ability to culture the cells in three dimensions.Secondly,A suitable hydrogel for the delivery of antigen(tumor cell lysate TCL)and TLR3 agonist(poly(I:C))was screened from four different molecular weight polypeptide hydrogels synthesized.By prolonged and sustained release of tumor-specific antigens and immunomodulators in situ,their ability to recruit and regulate DCs in vivo was investigated.The anti-tumor effect of this novel hydrogel vaccine was studied by a mouse melanoma treatment model and its mechanism of action was explored.Once again,An injectable self-assembled polyethylene glycol-poly(L-alanine)polypeptide hydrogel was selected for loading antigen(tumor cell lysate TCL),granulocyte-macrophage colony-stimulating factor(GM-CSF)and immune checkpoint inhibitors(ICIs).The ability to recruit and regulate DCs was investigated in vitro and in vivo.The anti-tumor effect of this novel hydrogel vaccine combination therapy strategy was studied through a mouse melanoma treatment model and its mechanism of action was also explored.At last,the peptide sequences containing the epitopes are prepared by attaching the epitopes to the self-assembled small molecule polypeptides by means of a solid phase synthesis technique.Polypeptide copolymers bonding epitopes in aqueous solution assemble into nanostructured solutions or hydrogel vaccines.The peptides that bind different epitopes are then co-assembled to prepare a multivalent epitope vaccine.The ability of the novel epitope vaccine to stimulate cellular immune responses at the cellular or animal level was evaluated.The safety and efficacy of tumor immunotherapy were evaluated to verify the superiority of the epitope vaccine.Results:First of all,a series of PEG-polypeptide copolymers were successfully prepared and self-assembled in aqueous solution to form nanostructures to obtain nano-aqueous solutions or hydrogels.The SEM results show that the hydrogel has a porous interpenetrating network structure.The CCK-8 was used to verify that the PEV copolymers have good cell compatibility.The three-dimensional cell culture results showed that the cells could survive and stably proliferate in the hydrogel.Secondly,an injectable polypeptide hydrogel vaccine formulation was successfully constructed that could simultaneously load antigens and immunomodulators.Peptide hydrogels are capable of sustained release TCL and poly(I:C)for more than one week,serving as an effective vaccine delivery vehicle.This vaccine formulation can recruit,activate and mature DCs in vitro and in vivo.The use of a polypeptide hydrogel delivery vehicle extends the duration of the antigen at the site of injection and promotes the percentage of antigen home to the lymph node.The results of immunization in B16 tumor-bearing mice showed that the polypeptide hydrogel vaccine formulations containing TCL and poly(I:C)could effectively induce activated DCs to migrate to the draining lymph nodes and initiate a strong CTL response to inhibit tumor growth.Once again,an injectable polypeptide hydrogel vaccine formulation that simultaneously loaded antigen and immune checkpoint inhibitors was successfully constructed.The hydrogel sustained release of TCL,GM-CSF and ICIs for up to 10 days.Hydrogel vaccines can effectively activate DCs and stimulate systemic CTL responses.Combined with immune checkpoint inhibitors,a stronger anti-melanoma therapeutic effect can be obtained.They can enhance the expansion of effector CD8+ T cells in the spleens and tumors of immunized mice,while decreasing the proportion of Treg cells in the spleens.At last,this chapter is based on more specific,higher safety and more efficient epitope as antigens.Preparare the nano structured solution or hydrogel epitope vaccine by chemical modification and self-assembly methods.The levels of IgG antibodies and IFN-?,TNF-?,IL-4,IL-2 cytokines produced in serum were significantly increased.The splenic CD4+T and CD8+ T cell populations proliferated significantly.The vaccine improves the immunogenicity of the epitope and enhances the specific immune response.Conclusion:The hydrogel vaccine we prepared can effectively recruit a large number of dendritic cells in vivo and promote their maturation,thereby activating tumor-specific cytotoxic T cell immune responses and killing tumor cells.Combination therapy with immune checkpoint inhibitors suppresses the immune microenvironment and significantly reduces the production of Tregs,thus further improving the anti-tumor effect.The design and construction of a bi-epitope hydrogel vaccine greatly enhances the immunogenicity of the epitope polypeptide,and can evoke even more intense cellular and humoral immune responses than the naked epitope and protein antigen.The research work of this dissertation shows that the injectable polypeptide hydrogel is an effective delivery vehicle of vaccine and immune checkpoint inhibitor.It can provide a novel carrier in the construction of tumor vaccines,providing potential therapeutic means for tumor immunotherapy.