Design And Immunological Studies Of Novel Hapten-modified Carbohydrate-based Tumor Vaccines

The aberrant glycans overexpressed on the cancer cell surface,known as tumor-associated carbohydrate antigens(TACAs),play important roles in tumor cell metastasis and signal transduction.TACAs are correlated with tumor progression and the poor prognosis of patients.Therefore,TACAs are attractive targets for the development of anti-cancer vaccines.However,TACAs have low immunogenicity,and most TACAs are T-cell-independent antigens.They do not bind with the major histocompatibility complexes(MHCs)directly and cannot activate T-cells by themselves.To overcome this obstacle,the conventional strategy is to conjugate TACAs to a carrier protein,which stimulate the body to produce specific antibodies for tumor immunotherapy.However,TACAs are human self-antigens,which can easily induce immune tolerance and immunosuppression,further increasing the difficulty of vaccine development.Therefore,augment the immunogenicity and break the immunotolerance of TACAs is one of the key issues in the development of tumor carbohydrate vaccines.Endogenous antibodies are naturally pre-existing antibodies in human serum,such as anti-2,4-dinitrophenyl group(DNP)and anti-rhamnose(Rha)antibodies,which account for 3-8%of human serum antibodied.This thesis uses the above two endogenous antibodies to enhance antigen presentation and mediate the ability of the immune system to kill target cells,and develop novel carbohydrate tumor vaccines and antitumor drugs based on endogenous hapten modifications.The main results are as follows:(1)The tumor-associated carbohydrate antigen GM3 was modified by DNP to augment the immunogenicity of TACA and enhance the ability of carbohydrate antigen presentation.DNP was modified to the C5 position of sialic acid at the end of GM3 antigen by chemical enzyme method,and then conjugated with KLH carrier protein to obtain GM3-NHDNP-KLH vaccine,with the loading of carbohydrate antigen of 6%.After DNP modified GM3 antigen was expressed on the surface of tumor cells by glyco-engineering,flow cytometry showed that antibody serum could recognize tumor cells modified by glyco-engineering.Complement dependent cytotoxicity experiment in vitro showed that antibody serum could mediate significant CDC effect on killing tumor cells through synergistic effect of anti-DNP antibody and anti-GM3-NHDNP antibody,with a cell lysis rate of 45.7%.(2)The weak immunogenic glycoprotein vaccine sTn-BSA was modified by rhamnose(Rha)multivalently,which enhanced the immunogenicity of TACA and reduce the immune response to the carrier protein.sTn and BSA proteins were conjugate by chemical synthesis method,and the loading of sTn antigen was 7.8%.Then,sTn-BSA was modified by Rha to obtain three vaccines with different loading of Rha haptens,namely,sTn-BSA-Rha₁,sTn-BSA-Rha₂ and sTn-BSA-Rha₅,and the loading of Rha was 0.5%,1%and 2.8%,respectively.In vivo immunological studies showed that in the mouse model with high titer of anti-Rha antibody,the higher the content of Rha hapten on the vaccine was,the more IgG antibodies were produced.Among them,the antibody titer produced by sTn-BSA-Rha₅ immunization was the highest,which was 64 times that of the control group.Flow cytometry and CDC experiments in vitro showed that the antibody serum could recognize the tumor cells expressing sTn antigen and mediate significant CDC effect to kill tumor cells.The cell lysis rate of sTn-BSA-Rha₅ was 57.3%.(3)Non-covalent glycoprotein conjugate vaccine was formed by self-assembly of multivalent Rha modified?-cyclodextrin(?-CD)and adamantane(Ada)modified sTn-BSA.It was found that multivalent Rha on?-CD could efficiently recruit endogenous anti-Rha antibodies to improve the immune activity of the vaccine and enhance the antigen presentation ability of sTn.Three kinds of non-covalent bond supramolecular vaccines with different PEG linker lengths were prepared by host-guest self-assembly method,which were sTn-BSA-Ada₆/CD-PEG₁-Rha₇,sTn-BSA-Ada₆/CD-PEG₃-Rha₇ and sTn-BSA-Ada₆/CD-PEG₆-Rha₇.In vivo immunological studies showed that the IgG antibody titer produced by non-covalent sTn-BSA-Ada₆/CD-PEG₆-Rha₇ vaccine was the highest in the high titer anti-Rha antibody mouse model,which was 9.5 times that of the control group.In the non-Rha mice model,the anti-Rha antibody produced in the early stage of immunization can play a role in self-enhancing immune response in the late stage of immunization.The IgG antibody titer produced by sTn-BSA-Ada₆/CD-PEG₆-Rha₇ immunization was 8.8 times higher than that of the control group.Flow cytometry and in vitro CDC experiments showed that all groups of antibody serum could recognize the tumor cells expressing sTn antigen and mediate CDC to kill cancer cells.(4)The ability to recruit antibodies and kill tumors was enhanced by multivalently modifying hyaluronic acid(a specific ligand that can recognize CD44)with hapten DNP.Nine kinds of multivalent antibody-recruiting glycopolymers(MARGs)modified by DNP were synthesized,which were HA-[PEG₁-DNP]₂,HA-[PEG₁-DNP]₄,HA-[PEG₁-DNP]₈,HA-[PEG₃-DNP]₂,HA-[PEG₃-DNP]₄,HA-[PEG₃-DNP]₈,HA-[PEG₆-DNP]₂,HA-[PEG₆-DNP]₄ and HA-[PEG₆-DNP]₈.Immunofluorescence and flow cytometry experiments showed that these constructs could specifically recruit anti-DNP antibodies to cancer cells expressing CD44,and the antibody recruitment ability was positively correlated with the modified DNP hapten.In vitro ADCC and CDC experiments showed that MARGs could kill target cells by mediating ADCC or CDC.Among them,HA-[PEG₃-DNP]₈ had the highest cytotoxicity with cell lysis rate of34.3%and 50%,respectively.The in vivo antitumor experiment of HA-[PEG₃-DNP]₈ showed that after treatment,compared with the control group,the tumor inhibition rate of HA-[PEG₃-DNP]₈ group reached 55%,which was 12 times that of HA group.