The Study On The Construction And The Immunological Activity Of The Tumor Vaccine Combined With CEA-epitopes And IL-18 In Microencapsulated Cells

Carcinoembryonic antigen,CEA, is an important tumor-associated antigen and tumormarkers being approved internationally. CEA positive tumors rank top among malignanttumors as for morbidity and mortality, however, we are not satisfied with the result ofsurgery, radiotherapy and chemical therapy. Recently, with the development of molecularimmunology and the technique of recombinant DNA, gene therapy has been a new way forthe therapy of malignant tumor. Particularly since Tang report the DNA vaccine (genevaccine) and then initiated the third revolution of vaccine, all kinds of tumor associatedantigen vaccine are developed one after another and be recognized as the most promising inmarket among all the therapeutic tools. CEA, belongs to tumor associated antigen, is of some antigenicity, and can berecognized by specific CTL in vivo and then induce immunological response to tumor.However, most research reports that most tumor sufferers manifest the status of immunesuppression. Therefore, we insert the gene of CEA into the eukaryotic expression andconstruct the gene vaccine with the technique of gene recombination. The vaccine canarouse the immunological response to CEA positive tumor and promote the extinction oftumor. There is a huge range of tumors which are CEA positive. The vaccine of CEA has thesatisfied prospect of clinical application by the reason that it can overcome the shortcomingof other tumor vaccines that is the response to only one simple kind of tumor. Nowadays,there are many kinds of CEA gene vaccine are developed by the laboratories in domesticand abroad. CEA gene vaccine has been taken in to the first and second stage of clinicalassay in abroad. Although the gene vaccine bring the expectation of therapy of malignanttumor, the clinical implication is still interfered by the reason that mutation which threatento the organism can happens in the transgenic cell and the expression of purpose gene is notunder efficient control, so the transgenic cell can lead the anti-graft rejection against thehost. Therefore, how to overcome the difficulties mentioned before and improve the safetyof gene vaccine, it always be f cell (HFCL) in domestic and prepare the microencapsulatedCEA gene vaccine. At the same time, we transfect the antigen peptide of CEA and IL-18 byencapsulating them into microcapsule and then produce the gene vaccine whichco-expresses CEA and IL-18. we also observe the expression of purpose gene in vivo andmice and the immune state of mice which carry tumor after inoculation of the cellstransfected with encapsulated CEA and the inhibition of tumor in vivo. We investigate thepossibility of therapy for CEA-positive tumors and establish the foundation for the furtherclinical application.HFCL, which can synthesizes and excretes many kinds of regulatory factors (cytokineand adhesion molecule) and base material (collegen and enzyme), is an essential ingredientin the hemopoietic miroenviroment in vivo. Most research has show, HFCL is easy toculture and amplify in vitro, and the supernatant of the medium can stimulate the growth ofgranular progenitor cells. HFCL, with which as the transfection vector, is an ideal target cellfor gene therapy and the topic of our research since the expression of aim protein in it isveryefficient.At present, all of the CEA gene vaccines are constructed with the complete sequencewhich is 3.0kb, although the fragment is so long the immunogenity of the coding product isnowt high correspondingly. In our experiment, through DNA synthesis, restriction enzymedigestion and ligation, we cut off the two repeat exons(I and II) and select the region(1.3kb)which include signal peptide, N-terminal, part III and terminal C) with the technique ofPCR using the complete sequence of CEAcDNA as template and DNA recombination. Afteranalysis of sequencing, the open reading frame of CEA after gene recombination is notaltered. we insert this sequence into the multiclone stie of the eucaryotic expression vectorpcDNA3 and construct the recombinatent eucarytic expression vector pc-CEA . we transfectthe expression vector into HFCL with lipofactin and screen the positive clone with G418selection CEA has integrated into the genome of HFCL after confirmation by PCR. CEAhas been transcribed and translated after analysis of RT-PCR and western blot. We obtainthe new CEA79 antigen protein which weigh 70kD and can response to CEA monocloneantibody. The recombinant plasmid pc-CEA and CEA70 can be used as vaccine for thetherapy of CEA-positive tumor. The expression of CEA antigen in HFCL is 12.72ng/ml byELISA, which is a little higher than the secretion in colon strain HT-29. Thus, we obtainedthe transgenic cell line HFCL-CEA which can express the immunogenic protein of CEAefficiently.recently, with the concept of the recognition of tumor antigen immunocompetenceepitopes by T lymphocyte being established, the immune therapy being centered by theantigen peptide vaccine recognized by T lymphocyte made a great achievement. However,the shortcomings of vaccine of T cell epitopes are: the synthesized peptide is prone to bedegraded by proteinase since the molecular weight is small and half life is short (onlyseveral minutes), so the implication is limited since it can not stimulate the immune systemconstantly. In our research, we found the cDNA fragment which encodes the T cell epitopes.We insert the complete gene which synthesized concatemer of CEA epitopes into multiclonesite of the eucaryotic expression vector pcDNA3 and construct the recombinatent eucaryoticexpression vector pc-CEA epitopes,pc-CEA epitopes/IL-18. we transfect pc-CEA epitopes,pc-CEA epitopes/IL-18 into HFCL with lipofactin and screen the positive clone with G418selection. Through the work mentions before, we obtained the transgenic cell lineHFCL-CEA epitopes,HFCL-CEA epitopes/IL-18 which are immunogenic and can expressthe immunogenic protein of CEA stably and efficiently. The epitopes gene vaccineconstructed by connecting the cDNA which code the CEA antigen peptide serially would bemore targeting and specific since it remove the interference of independent sequences. Itcan induce T lymphocyte to evolve into CTL constantly and efficiently and thus remove theimmune tolerance to tumor, so our project is innovative.Moreover, we transfect the encapsulated vaccine with the microcapsule to HFCL.Microcapsule is a selective permeable membrane which is synthesized by chitosan and highmolecular polymer of halobios-sodium polymannuronate. Nutrition and micromoleculesecreted by living cell in the sack can freely enter the sack through the pore on the sackwhile macromolecular such as antibody and immuocyte are stopped outside the sack. So thesack provides cells in it a natural screen for immuno protection by avoid rejection. Thetransplant cell can live in the host and secret therapeutic recombination protein for a longterm. At present, many kinds of tissue are successfully been transplanted with the techniqueof microcapsule, the immune isolation therapy with microcapsule has been put intoinvestigations in human being on a small scale. These successful attempt manifest anextensive prospect for application for people and provide the solid bases for therapy ofmalignant tumor with the technique of construction of microcapsulated and recombinantgene vaccine.After confirmation of culturing in vitro, microcapsule can sustain intact shape andstructure in the cultural environment in vitro for a long term and the cell in it can keepgrowing, proliferation and then secret CEA protein. Microcapsule still have goodmechanical strength and tenacity and then can change shape but not break under somepressure, it recovers immediately after the external force is withdrawn. Therefore, we cantransplant the cells which is transfected with micorcapsulated CEA gene by directlyinjection to the body and exact the gene therapy for tumor ex vivo.We found the microcapsule has good biocompatibility, some kind of mechanicalstrength and function of immune isolation after we transplant the cell vaccine thransfectedwith microencapsulated transgenic CEA into the belly of mouse. The cells in the sack cansurvive for 3-6 months, so it is not necessary to accompany the immunosuppressive agent tothe microencapsulation when we transplant it into the cell. The heterogenesis cell also canbe used as the resource for the donator, HFCL not only can express the purpose geneefficiently, but secret the extracellular matrix which has the function of support and allowthe cells in the sack to grow separately, so it is more suitable to the technique ofmicroencapulation than the tumor cell lines. The capsulae vacuus is asepsis and of noimmunogenicity according the immunological index. It would not influence the increasingof weight of the experiment mouse and not induce the transformation of lymphocyte inspleen either. However, the microencapsulated HFCL-CEA vaccine microencapsulatedHFCL-CEA epitopes and microencapsulated HFCL-CEA epitopes/IL-18 vaccine can inhibitthe growth of CEA positive tumor and extend the survival time of tumor-carrying mouse.The result of flow cytometry proved that the microencapsulated CEA vaccine andmicroencapsulated CEA microencapsulated HFCL-CEA epitopes /IL-18 vaccine canenhance the specific immune response to tumor. At the same time, all the results haveshowed that the immune effect of microencapsulated CEA microencapsulated HFCL-CEAepitopes /IL-18 vaccine is much stronger than the microencapsulated CEA vaccine. IL-18,which can strengthen the immune response of the vaccine, is an ideal kind ofimmunoadjuvant. The immunological effect of the microencapsulated gene vaccine is betterthan bare DNA vaccine.In the recent years, microcapsule vaccine develops very fast including microcapsuleand microcyst vaccine, the technique of production and method of dosage keeps beingimproved in overseas, while there are not reports about that till now. especially that themicrocapsule are used to capsule part of CEA gene and CEA epitopes /IL-18 and thentransfected into HFCL to produce the microencapsulated CEA gene vaccine for the therapyof malignant tumor is not reported whether domestic or oversea.