Construction And Functional Study Of Heat Shock Protein 65-HER2 Multi-Epitopes Fusion Protein

HER2 (Human Epidermis Growth Factor Receptor 2) is a member of theHER receptor family. The HER2 genes locate in the long arm ofchromosome17 (17q21) and encode a transmembraneous glycoprotein withintrinsic tyrosime kinase activity. The molecular weight of HER2 is 185KD.Up to now, the ligants for HER2 have not been identified. HER2 monomer hasno function, HER2 receptor activation occurs via receptor dimerization withother members of HER family and initiating signals that promote cellproliferation, survival, migration, adhesion and differentiation. HER2 isexpressed low level in various cell types in normal tissue and over-expressedin tumor tissues of 20-30% breast cancer, ovary cancer, gastric carcinoma andnon-small cell lung cancer. HER2 expression level is correlated withtumorgenesis, metastasis and prognosis. Down-regulation of HER2 expressioncan regerss HER2 over-expressing tumor and enhance the reactivity ofchemotherapy and radiotherapy. So HER2 is an efficient target for theimmunotherapy to HER2 expressing tumor. Some therapeutic drugs designedto target HER2 have been developed, in which Herceptin, a humanizedmonoclonal antibody, has been approved to use as a first line drug to treatpatients suffering from over-expressing HER2 cancer. Heat shock proteins (HSPs) are molecular chaperones, which can helpfolding, refolding and degrading proteins. The current studies showed thatHSPs played a considerable role in tumor immunity response: 1) HSPs canactivate the innate immunity system to play tumor inhibition effect. Forexample: HSPs can activate NK cell and Macrophage and enhance theactivities of them to lyse tumor. 2) HSPs can activate the adaptive immunitysystem to play anti-tumor effect: HSPs help the antigenic peptide to enter theantigen-presenting cell (APC) through specific receptor. After entering theAPC, HSP assists antigens to be processed and presented in MHC class Ⅰpathways. Thus, antigenic peptides can be co-expressed with MHC class Ⅰmolecules on DCs and consequently stimulate the generation of specificcytotoxic T lymphocyte(CTL). At the same time, HSPs like HSP60, HSP65,HSP70 can elicit increased expression of MHC and co-stimulatory molecularlike CD80, CD83, CD86, CD40 on the surface of APCs. These investigationssupport its potential use as molecular adjuvant in vaccine.Based on the principles above mentioned, we constructed a fusion geneconsists of the BCG heat shock protein (HSP) 65 gene and HER2 genes,which parts encode multiple CTL and Th epitopes, then we got the fusionproteins. The recombinant purifed BCG heat shock protein 65-HER2 fusionprotein, designated HSP6-HER2, was used to verify whether it has the activityto inhibit the growth of HER2-expressing tumor.The contents of this paper are as follows:1 The expression and purification of HSP65-HER2In order to construct the vaccine which target HER2 positive tumors, wefused genes encoding the HER2 multi-epitope peptides and BCG HSP65together to get the recombinant pET28a-HSP65-HER2 plasmid. The plasmidwas then transformed into BL21 bacteria. The bacteria were cultured andharvested after being induced by IPTG. Then the expressed proteinHSP65-HER2 was purified.1.1 The synthesis of HER2 multi-epitope peptide geneIn order to obtain the gene encoding the HER2 polypeptide, wedesigned 4 set of primers: primer 1 and 1',primer 2 and 2',primer 3 and 3',primer 4 and 4'.The target gene was amplified by 4 round PCR using theseprimers. The first round PCR product was generated by Primer 1 and 1' andthen used as the template for the second round PCR reaction. The PCR productof the second round was generated by Primer 2 and 2' and then used as thetemplate in the third round PCR reaction. Similarly, the PCR product of theforth round was synthesized using sequence 4 and 4' as primer and the PCRproduct of the third round as template, the PCR product of the forth round wegot was the gene encoding HER2 polypeptide.1.2 The construction of pET28a-HSP-HER2 plasmidThe synthesied HER2 gene was cloned into pMD-18T vector to getplasmid pMD-18T-HER2. HER2 DNA fragment was then subcloned intoexpression vector pET28a-HSP65 which has been constructed in our labbefore. The recombinant plasmid was identified as pET28a-HSP65-HER2 byelectrophoretic and sequence analysis.1.3 The expression and purification of HSP65-HER2The recombinant pET28a-HSP65-HER2 plasmid was transformed intoBL21 (DE3). A high-level expressing clone was selected and used to produceHSP65-HER2. Incubated cells were harvested after being induced by IPTG.The lysate of harvested cells was analyzed by using SDS-PAGE, showing a70KD band. The protein in the band can be recognized by anti-HSP65 andanti-histag monoclonal antibodies respectively. Recombinant HSP65-HER2 inthe induced cell lysate was purified to >95% purity through a metal ion chelateaffinity chromatography.1 The functional study of HSP65-HER2HSP65-HER2 was assigned some specific biological characteristics byour design at first: HSP can deliver antigen into the major histocompatibilitycomplex classⅠpresentation pathway of antigen-presenting cells (APC), andthus stimulate antigen-specific CD8+ T cell responses. Therefore,HSP65-HER2 can assist APC to present HER2-MHC Ⅰ complex toantigen-specific T cell, then the actived T cells will inhibit the growth ofHER2 positive tumor cells. Based on these principles, we will verify whetherHSP65-HER2 has tumor antagonism function as above confirmed by a seriesexpriments.1.1 Tumor inhibition effect of HSP65-HER2 in human tumor model in vitroTo investigate whether HSP65-HER2 can inhibit the growth of HER2positive tumor, we develop a tumor inhibition model in human in vitro firstly.2.1.1 The human tumor-inhibition model in vitroThe model was as follows: HLA-A2 and HER2 double positiveZR-75-30 and MCF-7 tumor cells were selected as target cells, CD8 positive Tcells induced by HSP65-HER2 loaded DCs were used as effectors, the targetsand effectors were co-cultured for 3-5 days and then the number of the targetswas detected.Dendritic cells (DCs) are professional APC and the only cells able toprime naive T cells. Consequently DCs were selected as APC in the study.Human mononuclear cells were isolated from buffy coats of healthy HLA-A2+volunteers by ficoll and percoll centrifugation and cultured in IMDMsupplemented with 200 U/ml GM-CSF and 200 U/ml IL-4 at 37 ℃, 5% CO2.On day 5, the immatured DCs were cultured with HSP65-HER2 for additional2 days. On day 7, the mature DCs were harvested.To obtain effectors that target to HER2 positive tumors, the autologousnaive CD8+T cells were isolated from PBMC and immunized withHSP65-HER2 loaded DC for three times at 7 days interval. In fact, as far asthe tumor inhibition model is concerned, the most important point is toidentify whether CD8+T cells exist in specific effectors relative to HER2peptide. Therefore, we will detect it at this point.DimerXI HLA-A2:Ig reagent was used to detect antigen-specific T cellsby a quantitative assay. In our experiment, we detected the HER2 specificCTLs using HER2 peptide P190-3 and P190-5 loaded Dimer. No peptideloaded Dimer was used as control. The result showed that: HER2 peptideP190-3 and P190-5 specific CD8+ T cells can be induced by HSP65-HER2loaded DCs.Based on above experiment, the model of tumor inhibition system invitro we construct is: co-culture HER2 positive tumor target cells andHSP65-HER2 loaded DCs induced CD8+ effectors for 3-5 days and thenobserve the growth of tumor cells.2.1.2 Tumor inhibition effect of HSP65-HER2 in human in vitroHLA-A2+ and HER2 expressing ZR-75-30 or MCF-7 tumor cells wereused as target cells. The cells above were inoculated into 96-well platesrespectively and co-cultured with the effectors generated by HSP65-HER2 orHSP65 pulsed DCs for 3-5 days. The number of target cell was counted in theend of experiment. The result indicated that the effectors generated fromHSP65-HER2 pulsed DCs can inhibit the growth of HER2 expressing tumorcell in vitro.2.1 The tumor inhibition mechanism study of HSP65-HER2To preferably define the tumor inhibition effect of recombinantHSP65-HER2, we approach relative mechanisms through a series ofexperiments. The result showed that: HSP65-HER2 inhibited the growth ofHER2 positive tumor cells by induced both specific and non-specific immuneeffect.2.2.1 The study of HSP65-HER2 specific tumor inhibition mechaniam2.2.1.1 The effect of HSP65-HER2 on immature dendritic cellsImmature dendritic cells (iDC) were derived from human PBMC byinduction with human IL-4 and GM-CSF for 5 days. HSP65-HER2 was addedinto iDC and incubated for another 2 days. Negative controls in thisexperiment were groups of medium, HSP65 and LPS. On day 7, cells indifferent groups were harvested. The expression of CD40, CD80, CD83, CD86,HLA-A2 on the surface of DCs was analyzed by flow cytometer. The resultrevealed that HSP65-HER2 and HSP65 could upregulate the expression ofCD40, CD80, CD83, CD86, HLA-A2 on the surface of DC. The up-regulationof CD86 was positively correlated with the dose of HSP65-HER2.2.2.1.2 Induction of human HER2 specific CTL by HSP65-HER2① Human CD8+ T cells induced by HSP65-HER2 loaded DC were used aseffectors, T2 cells or T2 cells loaded with 100μM HER2 peptides were used astarget cells. The CTL assays suggested that the mature DC, derived frommononuclear cell induced by HSP65-HER2, could induce and generate HER2specific CTL. The CTL could lyse T2 cells loaded with HER2 peptide.② Human CD8+ T cells induced by HSP65-HER2 loaded DC were used aseffectors. Human HLA-A2+ and HER2+ ZR-75-30 or MCF-7 tumor cell wereused as target cells. Other controls in this expriment were: CTLs induced andgenerated by DC pulsed no antigen (medium control) or pulsed with HSP65(HSP65 control). The CTL assays showed that HER2 specific CTL generatedby HSP65-HER2 pulsed DCs could lyse HER2 positive tumor cell ZR-75-30and MCF-7.2.2.1.3 The study of tumor apoptosis induced by effectors generated byHSP65-HER2HLA-A2 positive and HER2 expressing ZR-75-30 or MCF-7 tumorcells were stained with PKH-26 and incubated in 96-well plates at 37℃,5%CO2 for 12h and then used as target cells. CTLs generated by HSP65-HER2were used as effector cells. Effector cells and target cells were co-cultured at30:1 ratio for 2h. All the cells in the plate were stained with FITC-Annexin Vin the end. The result showed that HER2 positive tumor cells would occurapoptosis induced by CTLs generated by DCs pulsed with HSP65-HER2.Itdemonstrated that HER2 specific CTLs generated in our experiment lysedHER2 expressing tumor cell via inducing apoptosis of tumor cells.2.2.1.4 Detection of IFN-γ secreted by CD8+ T cells induced by HSP65-HER2Resting CD8+T cells do not normally produce cytokines. T cell can beactivated when exposed to the specific antigen and secret some cytokines likeIFN-γ. For this reason, the amount of IFN-γ can reflect the state of cellularimmunity to a great extent.The result of intracellular IFN-γ detection by flow cytometry indicatedthat there are more numbers of IFN-γ-secreting CD8+ T cells(antigen-specific T cell)in the CTLs induced by HSP65-HER2 pulsed DCswhen coltured with HER2 peptide loaded T2 cells than unloaded T2 cells. Inanother expriment, the result showed that there are more antigen-specific Tcells (IFN-γ-secreting CD8+ T cells) in CTLs induced by HSP65-HER2 pulsedDCs than CTLs generate by no antigen pulsed or HSP65 pulsed DCs whencoltured with HER2 expressing tumer cell MCF-7.2.2.2 The non-specific tumor inhibition mechanism study of HSP65-HER2From the result above, we got the information that effectors induced byHSP65 loaded DCs can lyse HER2 positive tumor cells to a certain extent. Wespeculated that the tumor inhibition effect induced by HSP65 is related with itsmolecular adjuvant characteristics. Experiments to define the non-specifictumor inhibition mechanism of HSP65-HER2 were performed.2.2.2.1 The activation effects of HSP65-HER2 on human PBMCHSP65 is an immune stimulator. We verify the immune stimulationeffect of HSP65-HER2 via analyzing whether the human PBMC will beactivated by co-culturing with HSP65-HER2 or not. The control group in thisexperiment is: medicum control, HSP65 control and LPS control. The resultshowed that CD8+T cells;CD56+NK cells;CD19+B cells and CD14+ cells inhuman PBMC could be activated after co-cultured with HSP65-HER2.2.2.2.2 The effect of HSP65-HER2 on activity of human NK cellsThe result above showed that NK cells could be activated byHSP65-HER2. In this part, we make sure that if HSP65-HER2 or other HSP65fusion proteins which constructed in our lab can enhance the anti-tumoractivity of human NK cells. PBMC co-cultured with HSP65-HER2 or otherHSP65 fusion proteins for 12h were used as effector cells. K562 cells, theNK-sensitive tumor cell, were used as target cells. The control groups in thisexperiment were: medium and LPS control. The result indicated that HSP65fusion proteins could enhance tumor lysis ability of NK cells.2.3 Effect of HSP65-HER2 in mice2.3.1 Preparation of GFP-HER2 transfected B16 cell lineThe pCDNA3-GFP-HER2 recombinant plasmid containing greenfluorescent protein (GFP) and HER2 epitopes fusion gene has beenconstructed and tansfected into B16 melanoma cells of C57BL/6 origin. Thetransfected cells were selected in G418 containing medium and identified byRT-PCR, fluorescence microscope and FACS assays, the selected transfectantswere named HER2+B162.3.2 Induction of HER2 specific CTL by HSP65-HER2 in miceC57BL/6 mice were divided into two groups randomly and immunizedwith 10μg recombinant protein HSP65-HER2 or 0.9%NaCl respectively onday 0, 14, 28. The mice were sacrificed on the fifth day after the thirdimmunization and the cells isolated from spleen were used as effector cells;GFP-HER2 transfected B16 cells were used as target cells. The result showedthat the CTLs in mice induced by HSP65-HER2 could lyse HER2 expressingtumor cells in vitro.In conclusion, we constructed a recombinant fusion protein,HSP65-HER2, which consists of BCG HSP65 part and multiple CTL and Thepitope peptides part derived from HER2. The biological effect of HSP65-HER2 was studied. The result showed that: Human CTLs generated byHSP65-HER2 loaded DCs can lyse and inhibit the growth of HER2 expressingtumor cell in vitro. Advanced study indicated that: HSP65-HER2 inhibited thegrowth of HER2 positive tumor cells by induce both specific and non-specificimmune effect. The primary researches in mice provide further evidences thatHSP65-HER2 can induce HER2 specific CTL in mice.The achievments in this project strongly indicate that HSP65-HER2 is animmunotherapeutic protein and will be applied to prevent and treat HER2+cancers in the near future.