Studies On Carbohydrate-Based Anti-Tumor Vaccines By Structural Modification Or Self-Adjuvanting Strategies

Tumor is often accompanied by changes in glycosylation.Tumor-associated carbohydrates antigens(TACAs)are widely expressed in a variety of tumor cells and are the preferred targets for the treatment and prevention of tumors.However,TACAs are easy to be recognized as‘self'by immune system,leading to immune tolerance.As a result,in this thesis we want to investigate the anti-tumor activities and immunological activities of the modified TACA conjugate vaccines and the constructed self-adjuvanting vaccines by the two strategies:the strategy of antigen structure modification to improve the immunogenicity of antigen,and the strategy of self-adjuvanting to promote immune response.The immunotherapy strategy based on cross-reactivity is to modify the natural sugar antigens.The induced antibodies not only recognize the modified antigens,but also recognize the natural antigens on the tumor cells,thereby improving the immunogenicity and breaking the immune tolerance.Therefore,in the first part of this thesis we study the immunological activity and antitumor activity of the modified TACAs.Adjuvants are thought to be a necessary feature to make cancer vaccines immunogenic enough to break immune tolerance to tumorantigens.The covalent coupling of both the antigen and the adjuvant to a single molecule can improve the involvementof antigen-presenting cells,avoid the use of highly toxic adjuvants,and still be able to elicit an effective immune response.Synthetic endogenous adjuvant vaccine is a good strategy to improve the immune response of anti-tumor sugar vaccine.Therefore,in the second part of this thesis we construct several self-adjuvanting vaccines and study their immunological activity.The specific contents and results of the study are as follows:In the first section,Tn and modified Tn antigens were covalently conjugated to carrier protein CRM197(Cross reactive material of diphtheria toxin)by reductive amination.The healthy mice were immunized with C34 adjuvant and the glycoconjugatesvaccines.The immunological results of these glycoconjugates indicated that N-fluoroacetyl-modified Tn-CRM197(S6-CRM197)elicited higher titers of antibodies which cross-reacted with native Tn antigen than the unmodified S2–CRM197 did.The IFN-?-producing frequency of lymphocytes in mice treated with S6–CRM197 was obviously increased,compared to that of mice vaccinated with S2–CRM197,which was typically associated with the Th1 response.Moreover,the elicited antisera against antigen S6–CRM197 reacted strongly with the Tn-positive tumor cells,implying the potential of this glycoconjugate as an anticancer vaccine.The potential application of fluoro modified for antitumor carbohydrate vaccines was verified on Tn antigen.Next,we studied the anti-tumor activity and mechanism of three fluorine-containing STn derivatives(Y2,Y3,Y4)in tumor-bearing mice.In this study,a mice lung metastatic tumor-bearing model was constructed with a murine colon cancer cell line CT-26for investigating the antitumor activity of the fluorine-modified vaccines and the mechanism of tumor immunotherapy of the vaccines.Our results showed that immunization with Y4-KLH(keyhole limpet hemocyanin)in combination with adjuvant could remarkably prolong the survival of tumor-bearing mouse and resulted in anobviously reduction in tumor burden of lungs compared with STn-KLH(Y1-KLH).Anti-tumor mechanism studies have shown:The vaccine Y4-KLH could induce strong anti-STn IgG antibodies capable of specifically recognizing STn-expressing tumor cells.Moreover,these antibodies were able to mediate complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity against STn-positive human tumor cells.The vaccine Y4-KLH could stimulate antigen-specific T lymphocytes to secrete IFN-?,provoke stronger cytotoxic T lymphocytes immune response.Y4-KLH produced a mixed Th1/Th2-type response and produced an earlier-stage Th1 immune response than Y1-KLH.Immunization with Y2-KLH could induce a stronger humoral immune response and a weaker cellular immune response compared with Y1-KLH;Immunization with Y3-KLH could induce a weak cellular immune response,but could not elicit a strong STn-specific antibody immune response.The vaccine Y4-KLH still showed a certain degree of anti-tumor effect without adjuvant or in the constructedmice axillary tumor-bearing model.Subsequently,CRM197 was used as carrier protein to study the immune response induced by Y4-CRM197 in healthy mice combined with different adjuvants.The results showed that immunization with Y4-CRM197 could improve the humoral immune response without adjuvant or C34 adjuvant.In the presence of Freund's adjuvant,immunization with Y4-CRM197 enhanced cellular and humoral immune responses.In the presence of C34 adjuvant,Y4-CRM197and Y1-CRM197immunized lung colon cancer mice,respectively.Although there was no significant difference in the antitumor effect between the two vaccines,but Y4-CRM197could improve the ability of spleen lymphocytes to secrete IFN-?,improve the Th1 immune response,while promoting humoral immunity response.In the second section,?-galactosylceramide(?GC)is a rapid activator of NKT cells,and MUC1 is a tumor-associated glycopeptide antigen.?GC was covalently coupled with MUC1for the first timeand immunological activity studies were performed.The results of immunological activity showed that?GC-MUC1 could activate DC cells and iNKT cells and release large amounts of IFN-?and IL-4;could increase CD4~+T cell content and increase CTL activity;could activate B cells,promote rapid and strong antigen-specific antibody response,produce a variety of antigen-specific IgG subtype;could promote the production of B cells that undergo high frequency mutations and the production of memory B cells.The oligonucleotide CPG1826 is an agonist of Toll like receptor 9(TLR9).Then,CPG1826 was covalently coupled with tumor-associated glycopeptide antigen MUC1.Immunological activity showed that MUC1-CPG1826 can also activate DC cells and iNKT cells;promote the activity of CTL and increase the content of CD4~+T cells in the spleen;can promote the body to produce rapid and strong antibody response;improve the contents of B cells in high frequency mutations and the contents of memory B cells.?GC and CPG1826 were covalently coupled with MUC1by us for the first time.The results of immunological activity showed that it could activate DC cells and iNKT cells,and promote the release of IFN-?and IL-4;could improve the activity of CTL and increase the content of CD4~+T cells in spleen,and promote the release of cytokine IFN-?in splenic lymphocytes;could promote the body to produce rapid antibody response,could activate B cells and improve the contents of B cells in high frequency mutations and the contents of memory B cells.In summary,in this thesis we systematically studied the anti-tumor activity of fluorine-modified STn and explored the anti-tumor mechanism.For the first time,the application of fluorine-modified STn as a candidate antitumor vaccine has been validated in the tumor model,which provides a candidate for the anti-tumor carbohydrate vaccine,which lays the foundation for the application of fluorine modified TACA as a candidate antitumor vaccine.?GC was first used in anti-tumor self-adjuvanting vaccine.It is also the first time that?GC and CPG1826 are covalently coupled with glycopeptide epitopes,providing a new idea for the study of anti-tumor vaccine strategy based on self-adjuvanting.