Construction And Anti-tumor Mechanism Study Of CTB Based Pentameric Peptide Vaccine

In recent years,with the advancement of immunological studies,and the interaction between tumor and the immune system has being constantly revealed,immunotherapy has become one of the most important antitumor treatment in addition to surgery and chemotherapy.Owing to the irstable and defined chemical structure,ease of preparation,low toxic side effects and low carcinogenic potential,peptide epitope-based vaccines have been one of major focus in genetic engineering of vaccine.A typical peptide vaccines possess tumor specific epitope and T helper(Th)epitope.Combined with adjuvant capable of enhancing cell mediated anti-tumor response,a peptide vaccine could induce tumor-specific immune responses,especially tumor-specific CTL(killer T cells).Many tumor associated antigens such as MUC1,CD20 and MAGE-3 are targets for the vaccine design.MUC1 is closely related to tumor growth,invasion and angiogenesis.In addition,abnormally high expression and abnormal glycosylated extracellular VNTR of MUC1 on tumor cell surface lead to exposure of epitope and thus become a particularly good tumor target for epitope vaccine design.Now many MUC1 VNTR peptide vaccines have been developed to control the MUC1-positive tumors.Most of these MUC1 peptide vaccines are constructed by conjugated or fused MUC1 VNTR peptide to Th epitope rich carrier protein with the method of chemical coupling or genetic engineering.But many peptide vaccines fail to induce MUC1 specific CTL in patients and thus did not show significant clinical benefit.Cholera toxin B subunit(CTB)can bind to TLR receptors and activate mucosal immunity,making it a good carrier protein or vaccine adjuvant.In addition,CTB can spontaneously form a non-covalent pentamers,which increase the size of the antigen,thus further enhancing its ability to induce immune response.In this study,we predicted the best B cell epitope on the surface of CTB by bioinformatic tool and replace it with MUC1 peptide to construct a new pentameric chimeric MUC1 peptide vaccine(CTB-MUC1).Combined with CpG plus Aluminum hydroxide,CTB-MUC1 can induce persistent MUC1 specific CTL and has significant anti-tumor effect.These results suggest that,CTB is a good carrier protein for cancer vaccine design.However,like most tumor peptide vaccine,CTB-MUC1 vaccine could not inhibit the tumor growth completely.This is manly due to the tumor induced immuno-suppression.To improve the therapeutic effect of immunotherapy,we should firstly remove the immunosuppressive factors,such as Myeloid derived suppressor cells(MDSC),regular T cells(Treg),tumor associated maccrophage(TAM)and they secreted inhibitory cytokines.Among these suppressor cells,MDSC is considered to be the main cause of cancer immunosuppression.By increasing accumulation in tumor patient,MDSC secrete many kinds of immunosuppressive factors to inhibit T cell activation and promote tumor angiogenesis.Therefore,removing tumor induced MDSC would be a very effective approach to change the tumor immune-environment and tore-activate anti-tumor immune responses.Calcium-binding protein MRP-8/14(S100A8 / S100A9)expressed by MDSC can be used as a surface marker for MDSC target drug development.In this study,we constructed pentameric CTB-S100A8/ CTB-S100A9 peptide vaccine using CTB as carrier protein.Animal experiment results showed that CTB-S100A8 / CTB-S100A9 can break self-immune tolerance to produce high titers of S100A8/S100A9 antibody while native S100A8/S100A9 protein did not produce antibody.Furthermore,tumor challenge result showed that CTB-S100A8/ CTB-S100A9 have significant tumor protective effect.The analysis of spleen and peripheral blood leukocyte from immunized mice showed that CTB-S100A8/CTB-S100A9 can reduce mononuclear and granulocytes source of MDSC in peripheral blood respectively.Furthermore,CTB-S100A9 also can reduce Treg in spleen and peripheral blood compared to other groups.These results showed that,CTB-S100A8/ CTB-S100A9 can effectively eliminate tumor immuno-suppression and enhance the anti-tumor immune response in tumor bearing mice.All the results above indicated that construction of pentameric peptide vaccine by epitope insertion or terminal fusion with CTB will be an effective vaccine design strategy applicable to other tumor-associated antigens.Moreover,CTB based pentameric peptide vaccines target to TAA and tumor immunosuppressive cells all can enhance anti-tumor immune-response in tumor bearing mice.So we should construct a multi-target peptide vaccine target to TAA and tumor microenvironment.This multi-target peptide vaccine may have more significant anti-tumor efficacy and greater clinical benifit.