Feasibility Evaluation Of Anti-tumor Vaccines Containing SLe~x And Activity Study Of SLe~x Derivative (sLe~x-ethyl Squarate)

Malignant tumors are one of the leading death causes among residents in China.Over the past 20 years,researchers have gradually focused their research on tumor-specific immunotherapy that minimizes toxicity,and made some progress.Immunotherapy mainly manifests its inhibitory effects on tumors in two aspects:first,it induces an effective anti-tumor immune response during tumorigenesis;second,it realizes long-term monitoring to tumors through immunological memory,and reduces the possibility of tumor recurrence.Immunotherapy will become one of the most important methods for cancer treatment.Tumor-associated carbohydrate antigens(TACAs)are present on both normal cells and tumor cells,but have a large difference in expression,and they are related to tumor metastasis and invasion.Sialyl Lewis x(sialyl Lewis x,sLex)is the ligand of the three adhesion molecules,E,P,and L-selectin,and is expressed on the surface of embryonic tissues,some immune cells and tumor cells.Studies have shown that sLex plays an important role in the invasion and metastasis of some malignant epithelial tumors.So sLex is a suitable target for the design of tumor vaccines,but sLex belongs to TACAs.TACAs cannot cause a T cell-dependent immune response,they do not produce high-affinity IgG antibodies,do not produce memory cells,and are easy to produce immune tolerance.Using a connexin carrier method to improve immunogenicity is a good solution which to convert a carbohydrate antigen from a non-T cell-dependent antigen into a T cell-dependent antigen.The CRM,97 protein is a diphtheria toxin(DTx)mutant,it losing DTs toxicity,but still retaining the same inflammatory and immunostimulatory properties.CRM197 widely used as a carbohydrate conjugate vaccine carrier.Keyhole limpet hemocyanin(KLH)is derived from the hemolymph of marine snails.Due to its large molecular mass and high complexity,KLH induces stronger immune responses than other carrier proteins.Moreover,since this protein is derived from molluscs,it is phylogenetically far away from mammalian species and is less likely to produce antibodies that cross-react with typical target samples during the assay.Therefore,KLH is widely used as a carrier protein.In this study,recombinant CRM197 and KLH were used as protein carriers and sLex was used as carbohydrate antigen.CRMi97-sLex and KLH-sLex conjugate vaccines were synthesized and evaluated.The activity of the sLex derivertive ligated to the protein carriers,sLex-ethyl squarate(sLexProSQ),was studied.The main research contents and results are as follows.Part One1 Expression and purification of CRM197 proteinThe CRM19-7 protein is expressed in large amounts in the form of inclusion bodies in E.coli and is also expressed in soluble form with small amounts.Considering the experimental amount,and the simplicity of protein purification and the stability of the purified protein,we mainly isolated and purified the soluble form.Under conditions of 20? 0.5 mmol/L isopropyl-?-D-thiogalactopyranoside(IPTG)inductionat,7.5 mg of purified CRM197 protein could be obtained from 1 L of bacteria medium.2 Synthesis and conjugating efficiency determination of the glyco-protein conjugate vaccineThe activited sLex,sLexProSQ,was coupled to CRM 197 protein,KLH protein,bovine serum albumin(BSA)and human serum albumin(HSA)under basic condations.The carbohydrate conjugate vaccines were analyzed by MALDI-TOF-MASS or sulfuric acid-phenol method.After calculation,the average carbohydrate content of one molecule of CRM 197 is about 4%;the average carbohydrate content of one molecule of KLH is about 7%;the average carbohydrate content of one molecule of BSA is about 3%;and the average carbohydrate content of one molecule of HSA is about 10.8%.3 Immunological evaluation of glycoprotein conjugate vaccineC57BL/6 mice were immunized with the synthetic CRM197-sLex and KLH-sLex conjugate vaccines and the antiserums were obtained.The antiserums were analyzed by ELISA for antibody titers(IgG).In the antibody titer analysis,the antiserum immunized with the CRM]97-sLex conjugate produced a higher antibody titer(1:72900),and the antiserum immunized with the KLH-sLex conjugate produced an antibody titer of 1:24300.The antigen-antibody complex mediated complement-dependent cytotoxicity(CDC)was determined by MTT assay.The antiserum produced in mice immunized with the conjugate vaccine produced a strong killing effect on human lung cancer A549 cells and murine melanoma B16-F10 cells in a dose-dependent manner in the presence of complement.The survival rate of B16-F19 cells co-incubated with 2 ?L antiserum in the presence of complement was approximately 40%.The survival rate of A549 cells co-incubated with 2 p,L antiserum was approximately 60%.The effect of antiserum on tumor migration was determined by cross-hatch experiments.The antiserum had a significant inhibitory effect on the migration of A549 cells at 36 h and 48 h.Anti-tumor experiments in vivo demonstrated that KLH-sLex conjugate had stronger effects on tumor inhibition and survival than PBS group,but was not as effective as KLH group.It is suspected that the activity of sLexProSQ in vivo may affect the tumor inhibition rate and survival of mice.Part Two1 Cytotoxicity assay of sLexProSQThe effect of different concentrations of sLexProSQ on the viability of EA.hy926 cells,B16-F10 cells,and A549 cells at different time was determined by MTT assay.The results showed that sLexpProSQ had no significant effect on cell viability at various concentrations(0.13 0.2,0.5,1,2,4,6,8,and 10 ?g/mL)and at all detection time points(6,12 and 24 h).2 Anti-E-selectin adhesion effect of sLexProSQThe effect of co-incubation of different concentrations of sLexProSQ with B16-F10 cells and A549 cells on the adhesion of E-selectin was determined by MTT assay.The results showed that sLexProSQ had a significant inhibitory effect on cell adhesion to E-selectin at a concentration of 800 ??mL.3.In vivo anti-tumor activity of sLexProSQSLexProSQ has a certain inhibitory effect on B16-F10 melanoma in high,medium and low doses.The average inhibition rate was 13%in the low-dose group,59.7%in the middle-dose group,and 27.4%in the high-dose group.The tumor tissue necrosis was observed by HE staining.The necrosis of the treated group was stronger than that of the PBS group.The abnormality of the tumor cells in the PBS group was more serious than that of the treated group.The immunohistochemistry results of CD34 showed that the neovascularization of tumor tissue in the treated group was less than that of the PBS group,and the neovascularization was minimal in the middle-dose group,consistent with the VEGFR-2 expression analyzed by Western blot.The effect of different doses of sLexProSQ on the levels of IL-6,TSNF-? and EL-1? was determined by ELISA.As the dose increased,the content of IL-6 decreased,and the content of TNF-? and IL-1? increased.Conclusion and significanceIn this study,CRM197-sLex and KLH-sLex conjugate vaccines were successfully synthesized and evaluated immunologically.The conjugate vaccine can stimulate C57BL/6 mice to produce antibodies with higher titers.The antiserum can kill tumor cells expressing the antigen and inhibit tumor migration in vitro,but did not produce a good anti-tumor effect in vivo.By evaluation of sLex derivative in vitro,sLexProSQ had been shown to have inhibitory effec on the adhesion of tumor cells to E-selectin,in vivo it had been shown to have antitumor activity.By studying the activity of sLex derivatives,in vitro studies have found that sLexProSQ can inhibit the adhesion of tumor cells to E-selectin,and in vivo studies found that sLexProSQ has a significant inhibitory effect on tumors at a certain dose.This study lays the foundation for the design and study of sLex-based vaccines,and also provides the expeimental basis for the development of sLex and its derivertives as anti-tumor agents.