Synthesis And Immunological Evaluations Of An Anti-tumor Vaccine:Sialyl Lewisx Tetrasaccharide And CRM197 Protein Conjugate

As is known to all,cancer has become one of the main killers to human health.Cancer immunotherapy has attracted more and more attention of the scientists because of its high efficiency,high specificity and long lasting memory effect.Significant progress has been made in traditional methods of cancer therapy during past decades,including chemotherapy,radio-therapy and surgical therapy.Built on the great success in vaccines against infectious diseases,cancer vaccines and immunotherapy have become attractive alternative approaches for cancer prevention and treatmentTumor-associated carbohydrate antigens(TACAs)are molecular markers overexpressed on human tumor cells,which can be used to distinguish tumor cells from normal cells.TACAs play important roles in tumor cell metastasis and signal transduction.Sialyl Lewisx(sLex)is a tetrasaccharide which is expressed on embryonic tissue and tumor cell surface.Studies showed that sLex is one of the ligands of E-selectin and it plays important roles in the invasion and metastasis process of some malignant epithelial tumors.SLex shows high affinity to C-type lectin in colon cancer cells.With the help of E-selectin.tumor cells overexpressing sLe'antigen can easily adhere to blood vessels and fall off from the primary tumor site to normal tissue.This process involves in cancer cell metastasis.Despite the great potential of TACAs-based antitumor vaccines as highly potent and specific cancer immunotherapy agents,they are still facing serious problems.Most TACAs are T-cell-independent antigens,so they do not bind with the major histocompatibility complexes(MHCs)directly and cannot activate T-cells by themselves.Covalently conjugating TACAs with protein or peptide carriers containing T-cell epitopes to convert them to T-cell-dependent antigens can remarkably improve their immunogenicity and the production of high-affinity IgG antibodies.CRM 197 protein is a variant of the diphtheria toxin(DTx,58 kDa)characterized by a single mutation(i.e.a glycine-glutamic acid substitution in position 52)that reduces its toxicity.The protein nonetheless retains the same immunogenic and in flammatory properties as the diphtheria toxin,and it is widely used as a safe carrier in conjugated vaccines.The main research contents and results are as follows1.Expression and purification of CRM197 proteinThe plasmid carrying CRM197 gene,pET22b-crm 197,was transformed into E coli BL21 by genetic engineering.The recombinant protein was expressed in the form of inclusion body.The inclusion body was washed and denatured by 8 mol/L urea solution.Soluble,stable,high purified and high yield CRM9 protein was obtained after renaturation.Induced with 1 mmol/L IPTC(isopropyl ?-D-1-thiogalactopyranoside)at 37?,55 mg of CRM197 protein could be obtained from 1L of bacteria medium2.Synthesis and conjugating efficiency determination of the glyco-protein conjugate vaccineThe conjugation of sLex tetrasaccharide to CRM197 or HSA protein was performed by "click chemistry" using squared acid as the linker.The glyco-protein conjugates were analyzed by MALDI-TOF-MASS.By calculation.it was found that each ISA protein molecule could conjugate 11.8 molecules of sLex tetrasaccharide,with the average glyco-loading efficiency of 13.1%and each CRM197 protein molecule could conjugate 13.3 molecules of sLex tetrasaccharide,with the glyco-loading efficiency of 16.0%3.Immunological evaluation of the glyco-protein conjugate vaccine.C57 mice were immunized by the synthesized CRM197-sLex conjugate vaccine,and the antisera was gained after immunization.Total antibody titer and antibody subtypes were analyzed by ELISA.The total antibody titer assay showed that CRM197-sLex conjugate induced high titer antibody(1:24300).The antibody subtype assay showed that antisera from mice immunized with CRM197-sLex conjugate consisted of mainly IgG subtype antibodies,especially IgG2c and IgG2b.CCK-8 assay was used to detect the complement-dependent cytotoxicity(CDC)of the antisera.The antisera induced by CRM197-sLex conjugate showed high cytotoxicity to human lung cancer cell line A549.Survival rate of the cells in the experimental group was 12.80%while control group incubated without complement was 97.88%and the group incubated with only complement was 74.23%ConclusionThe CRM,97-sLe' conjugate vaccine was synthesized,and its immunogenicity as a vaccine was evaluated.The conjugate vaccine showed strong immunogenicity and could stimulate the production of high titer of IgG antibody in C57 mice.The antisera from these mice showed high CDC on the cells expressing sLe" antigen.This thesis provides the basis for the glycoconjugate vaccine design based on sLex,as well as the basis for cancer immunotherapy using TACAs ans the gargets.