Functional Study Of HSP65-PSA Multiple-Epitopes Fusion Protein Vaccine

Prostate cancer is the most common type of cancer found in American middle-agedmen and the second leading cause of cancer death in men. Although the incidence andmortality of prostate cancer in our country is lower than that in western countries, theincidence rate has been gradually increased because of the environment pollution anddietary change. Surgery is the common but not the versatile treatment for prostate canceras most patients are in advanced stage when diagnosed and lose the opportunity ofsurgery. So general therapies such as hormone therapy and chemotherapy are veryimportant too. In addition, immunological therapy might be another option to treatprostate cancer. This article is about developing an effective recombinant protein vaccineagainst prostate cancer which will create wealth and value for society and the economy.Prostate specific antigen( PSA ), a kallikrein-like serine protease, is one of the mostimportant tissue specific tumor antigen expressed exclusively in normal prostate epithelialcells and overexpressed in prostate cancer cells. PSA in the blood is a very importantmarker in diagnosing and screening prostate cancer. The majority of prostate cancer cellsoverexpresses PSA than normal prostate gland cells, so PSA is a potential target for theinduction of T cell-directed immunotherapy against prostate cancer.Some epitope peptides that can be recognized by tumor-specific cytotoxic Tlymphocytes (CTL), play an important role in T lymphocytes mediated immune responseagainst prostate cancer. Some articles reported that antigens can be cleaved into shortpeptides by proteases, these peptides move to cell surface and bind to a Dimer of MHCclass I antigens and b2-microglobulin. The peptides are usually 8-9 amino acids in length.The resulting trimolecular complex could be detected by CTLs and cause T cells'activation as the first activation signal.Some epitope peptides that can be recognized by prostate tumor-specific CTLs hadbeen recently identified such as PSA oligoepitope peptide (designated PSA-OP), whichcontains motifs for HLA class I-A2, A3, A11 and B53 alleles. The potential advantages ofusing an oligoepitope peptide rather than a combination of single-epitope peptides astumor antigen are in generating cellular responses in a broader segment of the humanpopulation with different HLA-class I molecules and in activating the much morenumbers of PSA-specific precursors. Therefore , PSA-OP may be a potential candidatefor use in peptide-based vaccines for human prostate carcinoma.HSP65 are molecular chaperones which can help the tumor antigens when with themto enter the dendritic cells (DC). After entering the DCs, HSP65 assists antigens to beprocessed and presented in MHC class I pathways. Thus, antigenic peptides coupled orfused with HSP65 can be co-expressed with MHC class I molecules on DCs andconsequently stimulate the generation of specific CTL. Therefore, HSP65-tumor antigenfusion protein provides a new immunotherapy option to breaking immunotolerance ofhuman body against tumor antigen for recognition by CD8+ T cell.We expressed and purified a fusion protein named BCG HSP65-PSA multi-epitopepeptides fusion protein (in abbreviation, HSP65-PSA) from E.coli. In order to identifywhether it can be used for Prostate cancer therapy, we have tested whether it can generatePSA specific CTLs both in mice(in vivo) and in human(in vitro). We also have studiedthe possible mechanisms of HSP65 internalization. And to study the function anddetection of HSP65 fusion protein, we have prepared monoclone antibody against HSP65too.The main study results are divided into 5 parts as follows:1. Preparation of mouse tumor cell line stably expressing human PSA or MUC1To obtain effectively and rapidly mouse transfected cell, we developed a modifiedliposome transfection method which combind with physical transfected method. In briefly,it was mediated by dropper, limiting dilution cell clone technique and early detectiontechnique with confocal flourence microscope. The results show that efficiency of genetransfection by modified method with lipofectin was much higher than traditional method.The potential advantages of using modified rather traditional method are:① cells can beexactly quantitated and be directly diluted and cloned without cell amplification ordigested with trypsogen;② it can obtain stable transfected cell strain in a possible shorttime;③the method has high efficacy and do not need other equipment,and could beapplied in various kinds of laboratories.After B16, EL4 and RM1 cells had been transfected pCDNA3-GFP-PSA vector orpCDNA3-GFP-MUC1 vector by modified method, the expression of the GFP in thesecells' cytoplasm and nucleus can be observed under confocal microscope. Flowcytometric analysis show that the percentages of GFP proportion of EL4, B16 and RM1cell which had been transfected with pCDNA3-GFP-PSA plasmid are ranging from 69%to 83%, the EL4 cell has the highest percentage with 83%, RM1 cell has the lowestpercentage with 69%, in all three kinds of transfected cells. The percentages of GFPproportion of EL4, B16 and EMT6 cell been transfected with pCDNA3-GFP-MUC1range from 74% to 93%, and the EL4 cell has the highest percentage of GFP expressionwith 93%, EMT6 cell has the lowest percentage of GFP expression with 74% in all threekinds of transfected cells.To evaluate the expression of target protein in the transfected cell, which werecloned and selected by means of GFP detection, we used indirect immunofluorescencetechnique and western blot method. The indirect immunofluorescence results show thatPSA or MUC1 proteins are expressed both in cytoplasm and nucleu of these cells thesame as GFP distribition. The western blot result show that three kinds of transfected cellsall expression MUC1 protein. These results suggest that transfected cells after selected byGFP detection assay successfully expressed target protein.These three kinds of cell stably transfected pCDNA3-GFP-MUC1 play an importantrole in finishing pre-clinical research of HSP65-MUC1 recombination protein vaccinewhich have recently entried clinical trial.Recently, most of studies on transfection mainly focus on the preparation oftransfected agents and development of new methods, rather than study of transfected cellcharacteristics. In this study, to assess the persistence of high-level GFP expression ofEL4 and B16 cell transfected with pCDNA3-GFP-PSA or pCDNA3-GFP-MUC1 plasmid,we cultured these cells in IMDM complete medium without G418 and regularly observethe GFP by Flow cytometry. The results show that the GFP fluorescence stability is muchbetter in EL4 cells than B16 cell, and is better in cells transfected withpCDNA3-GFP-MUC1 plasmid than those transfected with pCDNA3-GFP-PSA plasmid.The percentage of GFP fluorescence gradually decreased along with time prolong.Therefore, we suggest that the stability of expression GFP protein in transfected cells isrelated to the cell type and the interest gene transfected.During flow cytometric detection, we found that the SSC of cell transfected withrecombination plasmid had been inserted interest gene had changed much more than thatof cells transfected with naked pCDNA3 plasmid as compared with cell untransfected.We also observed some granulo-fluorescence objects existing in the cytoplasm oftransfected cell by indirect immunofluorescence stain, and to make clear the reason ofsuch changes, we also observed the ultrastructure of B16 cell transfected withpCDNA3-GFP-PSA recombination plasmid or pCDNA3 naked plasmid. The results showthat there are many secretory vacuole in the cytoplasm besides proliferation mitochondriaand endoplasmic reticulum in pCDNA3-GFP-PSA transfected cells in contrast to nakedplasmid transfected cells. We suppose that the formation of secretory vacuole wererelated with the expression process of GFP-PSA recombination protein.In order to determine whether the expression of PSA or MUC1 gene would influencethe growth of transfected cells, we developed C57BL/6 mouse tumor bearing model bysubcutaneouly injecting EL4/PSA or EL4/MUC1 cells, and the wild type EL4 serve ascontrol cells. The results show that growth rates of EL4/MUC1 cell is faster markedlythan those of EL4/PSA or EL4 cells in the C57BL/6 mice (p≤0.01), but no differencebetween EL4/PSA and EL4. We can conclude that the growth of EL4/MUC1 cells isfaster than that of EL4 cell or EL4/PSA cells. But we don't know whether it is related tothe type of interest gene transferred into target cells by pCDNA3 vector with cationicliposomes, or other influence factor either. At the same time, we observed that some micedo not have tumor formation, we guess it was related to mouse individual specificities.2. The effect of HSP65-PSA recombinant protein inhibiting Tumor growthMouse prostate tumor cell line RM1 and mouse melanoma cell line B16 bothtransfected with human PSA epitope gene were used as targets in mouse model. B16/PSA or RM1/PSA were inoculated into C57BL/6 mice on day 0 and the mice wereinjected with HSP65-PSA protein with different dosages on day 0 and day 15. On day 15,the tumor nodule was palpated and tumor diameters were measured in two orthogonaldimensions and recorded daily. At the end point of the experiment, the mice weresacrificed and the tumor mass was weighted. It was observed that the tumor growth wasinhibited in mice after injecting HSP65-PSA compared to injecting PBS of both B16/PSA and RM/PSA cells. We also found that tumor growth in mice injected withHSP65-PSA was inhibited in both male and female and the tumor growth in male mice ismuch faster than in female mice.3. Development, identification and utilization of HSP65 McAbHSP65 has molecular chaperones function that takes part in antigen uptake,processing and presentation course. Some recombinant protein vaccines , which treattumor or virus infective disease using HSP65 as delivering platform, have been in thepreclinical stage of research. Therefore, the monoclonal antibody against HSP65 will playimportant role in study of mechanism, pharmacokinetics and side effect of these vaccine.We develop hybridoma cell secreting HSP65 monoclonal antibody with HSP65 andHSP65-PSA recombinant protein.The quantity and quality of mouse spleen cell immunized by antigen is veryimportant for obtaining high-level secretion antibody cell strain during developmentmonoclonal antibody. CpG ODN is a new type adjuvants that can induce proliferation ofalmost all B cells, we use CpG ODN/Alum mixture as adjuvants to immunize BALb/Cmice together with purified HSP65 recombination protein, the results show that the serumantibody level of mice immunized with CpG/Alum and HSP65 protein is obviouslyhigher than that of mice immunized with Freund/BCG and HSP65 protein after threebasic immunization.We obtained five strain hybridoma cell secreting monoclonal antibody againstHSP65 antigen after cell fusion and clone screening. The antibody titer are 1×103 inculture medium of these hybridoma cell and in 1×106～1×108 range in ascitic liquid.The specificity of these antibodies against HSP65 antigen was identified by wetern bolt.Subtypes of monoclonal antibody are mainly IgG1 and IgG2a identified by ELISA.There is competition effect between ⅠC1 and ⅠD6 orⅠC5, while there is nocompetition effect between other antibodies. These antibodies have been used to identifyand analyze of HSP65 fusion protein.CPG ODN 1826 is a new type adjuvant that can induce proliferation of B cell whenused as adjuvant for basic immunogen introduced into mouse by subcutaneously injection,and it can directly induce B lymphocyte activation especially by direct injection to spleen.Using CPG ODN 1826 as adjuvants to immunize mice during development monoclonalantibody have not been reported yet.4. Studies of HSP65 receptor on the surface of imDCDendritic cells (DCs) are professional antigen presenting cells (APCs) that playcritical roles in both innate and adaptive immunity. Some researches show that CD91molecular on the surface of DCs is the receptor of HSP gp96 and mediate theinternalization of gp96-polypeptide compound, but it is not clear if HSP65 fusion proteinbind to and internalize into DCs in a receptor-dependent manner. In the course of thisstudy we tried to use recombinant BCG HSP65 protein and purified DCs induced fromhuman monocytes in vitro for investigating the specific binds between protein and cellsand activation of DCs. In order to determine the specificity of HSP65 protein binding toDCs, we finish the binding test at 4℃ with fluorescence-labeled HSP65 and immatureDCs. The results show that HSP65 protein binding is specific and the percentage of DCsbinding fluorescence-HSP65 can gradually increase along with raising FITC-HSP65concentration, the curve show"S"shape. Furthermore, the results show that immature DCsfrom different donors have different percentage of binding fluorescence-HSP65 protein.In activation experiment, we found that HSP65 protein in 50ug/ml concentration canup-regulate expression of costimulating molecular on the imDC such as CD86 and havemuch more ability than the lipopolysaccharide (LPS) and cytokine cocktail on activatingDC.Since the CD91 molecular on DCs was suspected to be the receptor of HSP65, westudied the distribution of both HSP65 receptor and CD91 on the imDC by specificbinding test of FITC-HSP65 and indirectly immunofluorescence staining with anti-CD91and CY3-labled sheep anti-mouse antibody. We found that the distribution of CD91molecular is not related to the distribution of HSP65 receptor.We demonstrated that internalization of HSP65 protein at 37℃ is very fast, and hasachieved saturation in two minutes, the mean fluorescence value of imDC bindingFITC-HSP65 can increase gradually after prolonging reaction time. We discovered thatthe receptor-mediated FITC-HSP65 phagocytosis exist in the cytoplasm of DCs in theform of granular or massive, and couldn't be found in the nucleus of DCs. We alsodiscovered that imDC treated with 0.5mM H202 for 30 minutes at 37℃ or heat shock for30 minutes at 45℃ have obviously enhanced the ability of binding the FITC-HSP65, butthese treatments have no effect on the groups treated with HSP65 protein, INF-γor LPS.Our results highlight the important roles of receptors in modulating the function ofDCs, we further suggest that binding of imDC and HSP65 protein were uninfluenced byantibody against the HSP65.5. Studies of HSP65-PSA recombinant protein vaccine function in vitroDC are professional antigen presenting, a compelling body of evidence indicates thatthe initiation of immune responses requires activation of dendritic cells. In order toevaluate the function of HSP65-PSA to activate imDCs, we loaded the imDCs derivedfrom human PBMC with purified recombinant HSP65-PSA protein and induceautogenetic na?ve T cell to differentiate into cytotoxic CD8+ TC. We demonstrate thatHSP65-PSA can upregulate co-stimulatory molecules expression such as CD80, CD83,HLA-DR and HLA-A2 which is related with DCs activation, and induces DCs displaytypical characteristics of mature. It had been determined that optimal concentration ofHSP65-PSA inducing imDC to be mature is 50ug/ml. We also demonstrate that CD8+T-cells can be obtained from Na?ve TC by activating after three cycle with DC, which hadbeen loaded HSP65-PSA protein, and have detected CD8+ TC specific against PSAepitope peptide such as PA10-7, PA10-8 or PA10-9 by using HLA-A2:Ig dimer bindingassay. These results indicated that HSP65-PSA can activate DC and we couldsignificantly generate MHC class I restricted and PSA specific CTLs by peptide-pulsedDCs to activate na?ve TC in vitro.In order to analyze the frequency of T cells that secrete IFN-γ cytokine in responseto an antigenic stimulation, we cultured T cells with LNCaP cell or HSP65-PSAprotein in wells,The number of spots, which allows one to determine the frequency ofIFN-γ-secreting cells specific for a given antigen, was detected by ELISPOT assay. Ourresult indicated the groups which was activated with mature DC loaded HSP65-PSAprotein generated higher frequency of IFN-γ-secreteing cells than the groups activatedwith HSP65-PSA. In all of the recombinant protein groups, the Chaperon-PSA recallesCTL to generate higher frequency of IFNγ-secreting cells than HSP65-PSA or HSP65protein. And the above recombinant protein's activating CTLs to generate IFNγ-secreting cells is all higher than medium control group.In order to analyze the function of CD8+ T cells derived from na?ve TC activatedwith mature DC loaded HSP65-PSA protein, we used CD8+ T cells as effectors cells andT2 cells coated with human PSA epitope peptides as target cells. Using standard3H-release assay to study the specific cytotoxic effect by radioactive isotope 3H-TdRlabel technique and liquid scintillation counter. The results showed that the CD8+ T cellswhich were obtained from na?ve TC by immunizing with autologous DCs loadedHSP65-PSA in vitro exhibited a cytotoxic activity against T2 cells coated with PSAepitope peptides.We also detected the funtion of CD8+ T cells derived from na?ve TC by activatingwith mature DC loaded PSA epitope peptides. The results show that the CD8+ T cellsobtained after 3 cycle in vitro immunization of PSA epitope peptides loaded autologousDCs exhibited a cytotoxic activity against human prostate cancer LNCaP cell line.In summary, we have developed six kinds mouse cell strain that express human PSAor MUC1 epitope peptides by modified liposome transfection method and have identifiedcharacteristics of those transfected cells by confocal microscope, electron microscope,flow cytometry and western blot technique, as well as implantation test in vivo.We have expressed and purified recombinant HSP65-PSA protein as vaccine toinduce PSA specific CTL that can inhibit growth of PSA+ mouse tumor cells in vivo. Wehave prepared many monoclonal antibodies against HSP65 by hybridoma technique withCPG/Alum new-type adjuvant and recombinant HSP65 fusion protein, and have appliedantibody to study the function and receptor of HSP65 fusion protein.we have investigated that HSP65-PSA fusion protein can specifically bind to DCsand upregulate the expression of costimulating molecules on DCs, also can induce na?veTC to generate specific CTL with autologous matured DCs loaded by HSP65-PSA proteinor PSA epitope peptide. The CTLs induced can lysis the target cell such as T2 cellsloaded with PSA peptides or human prostate cancer cell.All above results strongly indicate that HSP65-PSA recombinant protein as aimmunotherapeutic proteins vaccine will be applied to prevent and treat prostate cancersin the near future.