Design,Preparation And Activity Evaluation Of Cholera Toxin-Like Anti-Tumor Chimeric Protein Adjuvant

Anti-tumor vaccines have received wide attention due to their ability to activate the autoimmune system to target cancer cells that express tumor-specific antigens.Because of their unique advantages,subunit vaccines derived from partial epitopes of tumor antigens could play a good role in cancer therapy.However,the weak immunogenicity of antigenic peptides and their tendency to be degraded often mediate tolerance and fail to elicit an effective cytotoxic T lymphocyte(CTL)response in vivo,making it difficult to achieve tumor therapy.The key to effectively exerting the anti-tumor effect of vaccines is to break the immune tolerance and activate the CTL response.Prostate cancer is considered to be a dominant target for studying tumor vaccines due to the presence of multiple specific tumor-associated antigens(TAAs)in its microenvironment and its relatively slow growth kinetics.As exogenous antigens,TAAs of prostate cancer need to be recognized by antigen-presenting cells(APC)and cross-presented to activate CTL by MHC class I restriction pathway.Adjuvants can increase the immunogenicity of antigens and help antigen presentation.However,standard non-conjugated adjuvant-peptide mixtures do not ensure that the two are co-localized to the same APCs.Therefore,in this study,the murine granulocyte-macrophage colony-stimulating factor(mGM-CSF)was fused to the N-terminus of A2 subunit of cholera toxin(CTA2)by genetic engineering methods based on the structure of the complete cholera toxin(CT)hexamer.The human-derived prostate-specific membrane antigen(PSMA)epitope peptides fragment PSMA624-632 was fused at the C-terminus of cholera toxin B subunit(CTB).CT-like chimeric proteins were prepared and its anti-prostate cancer activity and mechanism were studied.Methods: 1.Obtainment of CT-like chimeric protein.The expression vector was constructed,and the fusion proteins mGM-CSF-CTA2 and CTB-PSMA624-632 were expressed in the form of inclusion bodies by E.coli prokaryotic expression system,purified by column chromatography and assembled into hexamer chimeric proteins with citrate-tris base in vitro.2.Determination of the biological activity of CT-like chimeric proteins.The bone marrow proliferation assay and the GM1-ELISA assay were used to verify whether the chimeric protein possesses both mGM-CSF and CTB activities.3.Construction of human PSMA expressed mouse prostatecancer cells RM-1-PSMA-EGFP.The enhanced green fluorescent protein(EGFP)was used as a reporter gene to construct a vector expressing human PSMA.The mouse prostate cancer cell line RM-1 was transfected with plasmid pEF1-myc-his-PSMA-EGFP,and the transfection was detected by inverted fluorescence microscopy and laser confocal microscopy.4.Anti-tumor activity of CT-like chimeric proteins study.C57BL/6J mice were intranasally immunized with CT-like chimeric protein for five times,and the tumors were transplanted.The tumor growth and the life quality of the mice were observed,then the serum levels of cytokine IFN-? were determined.5.CT-like chimeric proteins help enhancing the immunogenicity of PSMA624-632 peptide.After immunization,the spleen lymphocytes were isolated and stimulated with PSMA624-632 peptide in vitro to observe the induction proliferation ability of CT-like chimeric protein.6.The effect of CT-like chimeric proteins on the induction of DC cell maturation.Bone marrow-derived DC cells were prepared and induced by CT-like chimera protein in vitro.The mechanism of immune activation was explored by morphological and flow cytometry to detect the proportion of maturity and phenotype.7.The effect of chimeric proteins on the activation of CTL cells was studied.The mature DCs were transferred back to C57BL/6J mice,and the spleen T cells were purified and induced into antigen-specific CTLs.The cytotoxicity of effector cells was detected by LDH kit,and finally the level of cytokine IFN-? in culture supernatant was determined.Results: 1.Native-PAGE and circular dichroism confirmed the successful preparation of CT-like chimeric protein.2.CT-like chimera proteins have the same ability to stimulate myeloid cell proliferation as commercial mGM-CSF.In addition,it exhibited a better ability to bind GM1 at low dose concentrations compared to the CTB-PSMA624-632 pentameric protein.3.The plasmid expressing human PSMA is efficiently transfected into RM-1 cells by liposome and PSMA-EGFP was highly expressed on the cell membrane.4.In tumor-bearing mice immunized with CT-like chimeric protein,the growth of tumors was significantly slowed down,and the level of IFN-? in serum was higher than control groups.The response of mouse spleen T cells immunized with CT-like chimeric protein to the same antigenic peptide PSMA624-632 was significantly enhanced,demonstrating that the CT-like chimeric protein enhances the immunogenicity of the antigenic peptide PSMA624-632.6.DCs stimulated by CT-like chimeric protein expressed a high level of co-stimulatory factor CD86 on the surface,which was significantly higher than that of the mixed protein group,indicating that the chimeric protein can effectively activate the maturation of DC cells.7.CT-like chimeric protein can enhance the ability of CTL to kill target cells.The level of IFN-? in the culture supernatant is higher than control groups.Conclusions: The CT-like chimeric protein designed and prepared in this study can significantly improve the immunogenicity of the tumor antigen epitope peptide and enhance its ability to induce DC cell maturation and activate CTL cells.This provides a new strategy for the adjuvant developments of tumor subunit vaccine.The mouse prostate cancer cells expressing the human PSMA protein can be used for screening anti-tumor vaccine activity.