Research On Anti-Tumor Effect And Mechanism Of A Novel Anti-Gastrin Vaccine

The gastrins(GAS),including amidated gastrin 34,glycine-extend gastrin 34,amidated gastrin 17,glycine-extend gastrin 17 and the precursor progastrin(proG),are all known peptide hormones secreted by G cells in antrum of stomach and mucosal membrane.GAS can promote gastric acid secretion and gastrointestinal mucosa growth by binding to their corresponding receptors;however,excessive production of gastrin 17s(G17s)and proG can promote digestive tumors(including gastric,colon,pancreatic and esophageal cancers)via autocrine,paracrine or endocrine mechanisms.Additionally,for these digestive malignancies,existing chemotherapeutics have low specificity,a significant number of toxic side effects and poor therapeutic outcomes(e.g.,short survival times,generally poor quality of life,and high mortalities).Thus,there is a great deal of clinical interest in developing novel therapeutic products and methods for these digestive cancers that would have good specificity,less toxic side effects and potent anti-tumour efficacy.The signal transduction pathways posited for the promotion of tumors by G17 s and proG are both numerous and complex,but targeting them has become an investigative strategy for the treatment of related digestive tumors by anti-gastrin therapies,which specifically include endocrine-secretion inhibitors,receptor antagonism,and anti-gastrin antibody methods.Endocrine-secretion inhibition(e.g.by somatostatin and antisense oligonucleotides)and receptor antagonism(e.g.by cholecystokinin receptor antagonists)are methods that have often had low anti-gastrin specificity,weak anti-tumor effects,and necessarily large dosages,with a significant number of toxic side effects.Anti-gastrin antibody methods include both passive and active immunizations.Passive immunizations,e.g.,anti-gastrin polyclonal or monoclonal antibodies,results in intermittent neutralization,requiring long-term infusions that are quite expensive and are often associated with unwanted immunogenic side-reactions caused by heterogeneous antibodies.The active immunization method has been achieved clinically by the application of vaccine-like immunogens,prepared with the epitope of G17 s.The use of targeted vaccines in cancer therapy has become a hot research topic of late,as it requires fewer injections and induces specific neutralizing antibodies.Some targeted vaccines have potent anti-tumour effects and thus have good prospects for clinical application.However,currently it can only specifically offset G17s' effects on promoting tumors which also can be promoted by proG directly or indirectly in most cases.Focusing on the problem,this project designed and synthesized a new vaccine which was composed of the common amino-terminal portion of G17 s and the common carboxy-terminal portion of proG all covalently linked to tetanus toxoid(TT)by specific peptide spacers.Our aim was to develop a novel anti-gastrins vaccine with the specific capability of neutralizing G17 s and proG,would have significant potent anti-tumour effects as well as minor toxic side effects.We studied the vaccine's pharmacodynamics effects including:(1)the preparation and identification of vaccine;(2)the evaluation of vaccine's effects of immune response and preliminary safety;(3)the anti-tumor effect and mechanism of antibody induced by the vaccine and its combination with chemical drugs in vitro;(4)the anti-tumor effect and mechanism of antibody induced by the vaccine and its combination with chemical drugs in vivo.The synthesized antigenic epitopes of G17 s and proG were linked to TT by eMCS respectively.The coupling rates of antigenic epitopes were detected by HPLC.The coupling products were respectively purified by dialysis and column chromatography,then were equally mixed to prepare GAS-TT.GAS-TT's capability of specifically binding TT antitoxin and corresponding anti-gastrins antibody,molecular weight were identified by immune double diffusion method,ELISA,MALDI-TOF MS respectively.Formulation of GAS-TT with AS adjuvant was prepared by high speed dispersed homogeneous method.The agent's pH,purity,bacterial endotoxin,abnormal toxicity and allergic reaction were detected.The results showed that the coupling rates of antigenic episodes were 5.6%(G17s),6.1%(proG)respectively;the molecular weight of GAS-TT was about 158000~166000 Da,which had good specifity to bind TT antitoxin and corresponding anti-gastrins antibody;the agent's pH,purity,content of bacterial endotoxin were 6.4,98.7%,95 EU/mg respectively,which also had no abnormal toxicity and allergic reaction.The vaccine was used to immunize rabbits and BALB/c mice,the specific IgG antibody titers in the serum of rabbits and mice,IFN-? secreted by the spleen cells of mice,the specific binding between antibody induced by the vaccine and antigens were identified by ELISA,ELISPOT and competitive ELISA respectively.Record and detect the immuned animals' general clinical manifestations,weight,hematology,serum biochemistry,the relative weight of main organs,pathology changes of main organs and injection sites.The results showed that the vaccine could effectively induce the production of specific neutralizing antibody and IFN-?;TT as protein carrier can effectively enhance the humoral and cellular immune response of antigenic epitopes,however the vaccine's adjuvant had no obvious immune response;animals' body weight increased significantly after immunization(P<0.01);the hematology and serum biochemistry of immuned mice were generally normal,whose main organs also had no obvious histopathological changes.The tumor cell lines,which can secrete gastrin and express corresponding receptor,were screened by Western blot.The pro-tumor effect of gastrins,anti-tumor effect of anti-gastrins antibody and its combination with chemotherapeutics were identified by MTT.The anti-tumor mechanism of anti-gastrins antibody including its effect on the expression of tumor cell apoptosis proteins(e.g.Bax,p53,Bcl-2),multidrug resistence associated proteins(e.g.MDR1,MRP1,GST-?,TS)in the cell lines was identified by Western blot.The results showed that tumor cell lines such as SGC7901,BGC823,SW1990 could secrete gastrin and express corresponding receptor;gastrins could promote the growth of these cell lines;the antibody could inhibit their proliferation and promote their sensitivity to chemotherapeutics(e.g.cisplatin,fluorouracil,gemcitabine,paclitaxel)by inhibiting the expression of multi-drug resistance proteins(e.g.MDR1,MRP1,GST-?,TS)in those cell lines.The tumors were implanted subcutaneously into the backside of BALB/c nude mice.The relative tumor proliferation and tumor weight inhibition were used to evaluate the therapeutic effect of antibody and its combination with lowered dosage chemotherapeutics.Record and detect the treated mice's general clinical manifestations,weight,hematology,serum biochemistry,the relative weight and pathology changes of main organs.The anti-tumor mechanism of anti-gastrins antibody including its effect on the expression of gastrins,multidrug resistence associated proteins(e.g.MDR1,GST-?)in the tumors was identified by immunohistochemistry.The results showed that the relative tumor proliferation and tumor weight inhibition of the combination treatment were <40%,>30% respectively,which corresponded to that of the treatment by using standard high-dose chemotherapy alone;the mice treated with standard high-dose chemotherapy alone exhibited abnormal clinical reactions(e.g.diarrhea,hypothermia),had body weights significantly decreased,white blood cells and platelets significantly reduced,total protein in the serum significantly increased;the liver and kidney of the mice treated with standard high-dose chemotherapy alone also had obvious histopathological changes such as patch necrosis associated with inflammatory cells infiltration in the liver,swelling degeneration and atrophy of the renal tubule epithelial cells in the kidney,however the mice treated with combination antibody-drug treatment had no those toxic side effects;the antibody also could promote the tumors' sensitivity to chemotherapeutics(e.g.cisplatin,fluorouracil,gemcitabine,paclitaxel)by inhibiting the expression of gastrins and multi-drug resistance proteins(e.g.MDR1,GST-?)in the tumors.In short,this study manifests that the novel vaccine has good immunogenicity,antigenicity and preliminary safety.Its combination with lowered dosage chemotherapeutics has synergistic anti-tumor effects,also can reduce toxic side effects and improve therapeutic effects.The study provides novel method and experimental evidence for developing muti-targets vaccine and treatment with improved therapeutic effects.