BCG Heat Shock Protein65-HER2 Multiple-Epitopes Fusion Protein

HER2(Human Epidermis Growth Factor Receptor 2,HER2),a 185KDglycoprotein,is a member of the ErbB receptor family. The HER2 hasessential function in the animal developing process and the cell cycle. Themice lacking the HER2 gene will die at the embryo period duringdevelopment.HER2 is expressed low level in various cell types in normal body andover-expressed in 20-30% breast cancer patients'tumor tissues, in ovarycancer, gastric carcinoma and non-small cell lung cancer. Theoverexpression of HER2 is negatively correlated with the expression ofestrogen receptor (ER). The patients over-expressing high level HER2 isinsensitive to the anti-estrogen therapy. The over-expression of HER2 iscorrelated with lower responses to various anti-tumor drugs and shorterDFS(Disease Free Survival ) or OS (Overall Survival ). HER2over-expression is a prediction factor of bad prognosis. Down-regulation ofHER2 expression can regress the HER2 over-expressing tumor and enhancethe attractive of drug therapy and radiotherapy.HER2 has been demonstrated as an efficient target for theimmunotherapy to HER2 over-expressing tumor. A humanized monoclonalantibody, "Herceptin", is a commercialized product targeting the HER2molecule. Herceptin can bind the HER2 efficiently and block the signaltransduction of HER2 heterodimers or homodimers. Herceptin has beenafforded to use as a first line drug to treat advanced breast cancer patientsover-expressing HER2.Heat shock proteins (HSPs) are molecular chaperones, which can helpother protein in folding and degradation. As a danger signal, HSPs may actas a bridge between innate immunity and the acquired immunity. Theresearches have shown that the HSPs play an important role in immuneresponse. HSPs can help the antigenic peptide enter the dendritic cell (DC)through specific receptor CD91 or TLR2/TLR4 and elicit signal transductions.After entering the APC, HSP assists antigens to be processed and presentedin MHC class I pathways. Thus, antigenic peptides either fused or associatedto/with HSP can be co-expressed with MHC class I molecules on DCs andconsequently stimulate the generation of specific cytotoxic T lymphocyte(CTL). In the presented project, we constructed BCG HSP65-HER2 derivedpeptide encoding gene and expressed BCG HSP65-HER2 derived peptidefusion protein in E.coli. After multiple step-purifications, the recombinant BCGHSP65-HER2 derived peptide fusion protein, named HSP-HER2ME, wereused to verify whether it could generate HER2 specific CTL both in mice andin human and elicit a growth inhibition of HER-2 expressing tumor in mice. 1.Obtaining the HSP-HER2ME fusion protein: Multiple-epitope-HER2 gene (HER2ME) encoded 4 CTL epitopesderived from HER2 was synthesized by 4 round-PCR and sequenced. Themultiple-epitope-HER2 gene was inserted to the C terminal of the BCGHSP65 gene in the expression vector pET28a-HSP65 to create a heat shockprotein-65-Multi-epitope-HER2 fusion gene, HSP-HER2ME gene. The fusiongene encodes a peptide consisting of 671 amino acids. The expressionplasmid pET28a-HSP65-HER2ME was transformed into BL21(DE3) bacteria.The transformed bacteria was cultured, induced by IPTG for 3 hours and thenharvested. The HSP-HER2ME fusion protein was isolated from the bacteriaand purified. The purified recombinant HSP-HER2ME, showed a single bandof 71KD in SDS-PAGE, was suited for the functional tests. 2.Obtaining Chaperonin10-HER2ME fusion protein Chaperonin 10 is a member of heat shock protein family. We fusedMultiple-epitope-HER2 gene to the C terminal of chaperonin 10 to haveproduced a chaperonin 10-Multiple-epitope-HER2 fusion gene. The fusiongene was cloned into expression vector pET28a and sequenced. Therecombinant plasmid was transformed into BL21 (DE3) bacteria. Therecombinant chaperonin 10-Multiple-epitope-HER2 fusion protein wasisolated from the transformed bacteria induced by IPTG for 3 hours andshowed a 24KD protein in SDS-PAGE. 3.Obtaining TAT-HER2 fusion protein The HER2ME gene was inserted to the downstream of the TAT gene inthe expression vector pET28. The expression plasmid was transformed intoBL21(DE3) bacteria. The recombinant TAT-HER2 fusion protein was purifiedfrom transformed bacteria for functional analysis. 4. Preparation of HER2ME transfected B-16 cell line To analyze the therapeutic effects of HSP-HER2ME, we establishedHER2ME gene tranfected B16 tumor cells. The transfected cells wereselected in G418 containing medium and identified by RT-PCR and Westernblot assays. 5. Effect of HSP-HER2ME on the induction of HER2 specific CTL and ofanti-tumor activity 1). Induction of HER2 specific CTL by HSP-HER2ME in mice 8-10 weeks old C57BL/6 male mice were immunized subcutaneouslywith recombinant HSP-HER2ME at various dosages on day 0, 14, 28. For thecontrol group, the same volume of PBS was administered to the mice. On thefifth day after the last immunization, the mice were sacrificed. Single cellsuspensions were prepared from spleen and lymph nodes of the mice todetect the HER2ME specific CTL. The results showed that HSP-HER2MEstimulated the generation of HER-2 specific CTL in mice. 2).Growth Inhibition of TAT-HER2ME loaded tumor cells induced byHSP-HER2ME in mice C57/BL6 mice were immunized with recombinant HSP-HER2MEsubcutaneously. B16 cells pulsed with TAT-HER2 were inoculated into onehind leg, the B16 cells without pulsing was inoculated into the other hind leg.On the 15th day after the tumor cell-inoculation, the mice were sacrificed.Tumors were isolated and weighed. The data showed that B16 cells pulsedwith TAT-HER2ME didn't develop into tumors in mice immunized withHSP-HER2ME, whereas B16 tumor cells not loaded with TAT-HER2MEdeveloped into tumors intensively. Immunization with HSP-HER2ME caninhibit growth of B16 cell pulsed with TAT-HER2ME in mice. 3) Growth Inhibition of B16 cells transfected with HER2ME geneinduced by HSP-HER2ME in mice C57/BL6 mice were subcutaneously immunized with HSP-HER2MEfusion protein (0.2-5μg/per mice), the control mice received same volume ofPBS. Five days after the last immunization, B16/HER2 tumor cells(1×105/mice) were inoculated into back of the mice. After the first day whentumors on the back of the mice were palpable, the tumor size was measureddaily. Tumor growth curve was drawn using Tumor area (㎝2) vs Time(day).The diagram showed that B16/HER2 tumor cell grew in the mice receivedHSP-HER2ME more slowly than in the mice received PBS. Immunizationwith HSP-HER2ME fusion protein could prolong the survival of mice bearingHER2-positive tumors. 6. Biological effects of HSP-HER2ME on human immune cells It is very difficult to study the biological effects of HSP-HER2ME inhuman being in pre-clinical stage. We studied the effects of HSP-HER2MEon human PBMC, DC and CTL. 1) Activation of PBMC by HSP-HER2ME The activation of human PBMC by HSP-HER2ME was studied firstly.The results showed that the HSP-HER2ME could up-regulate the expressionof CD25, HLA-A2 at various dosages. CD25, IL-2 receptor, was expressedonly on the surface of activated T and B cell. The up-regulation of the CD25indicated the activation of the PBMC; HLA-A2 can bind and present type Iantigenic peptides and its up-regulation indicated the activation of the APC. 2) Stimulation of DC by HSP-HER2ME The HLA-A2 positive PBMC were co-cultured with GM-CSF and IL-4 todifferentiate into immature dendritic cell (iDC). The HSP-HER2ME was usedto stimulate the iDCs to mature. The surface markers of the DC weredetected by FACS method. The results revealed that HSP-HER2ME coulddown-regulate the CD14 (receptor of the LPS) expression of DCs, upregulatethe expression of CD40, CD54(ICAM-I), CD80, CD83, CD86, HLA-A2,HLA-DR on the surface of DC. The cell displayed typical characteristics ofmature DC after induction by HSP-HER2ME. 3) Induction and generation of human HER2 specific CTL byHSP-HER2ME Human HLA-A2+ CD8+T cells were immunized in vitro with matureautologous DC induced by HSP-HER2ME to generate the HER2 specificCTL. The CTL were used as effector cells. T2 cells loaded with 10μM ofHER2 peptides were used as targeted cells. The CTL assays suggested thatthe mature DC, which is derived from mononuclear cell induced byHSP-HER2ME, could induce and generate HER2 specific CTL. The CTL canlyse the T2 cell loaded with HER2 peptides. In summary, we first fused the BCG HSP65 gene to amultiple-epitope-her2 gene encoding 3 HLA-A2 CTL epitopes and 1 HLA-A3CTL epitope derived from human HER2 to form a fusion gene named asHSP-HER2ME gene. The recombinant HSP-HER2ME fusion protein wasprepared from engineered E.coli. The fusion protein was tested in C57BL/6mice in vitro and in vivo. It was demonstrated that HSP-HER2ME could elicitHER2ME specific CTL and prohibit the HER2ME expressing tumor growth inmice. In human in vitro test, the HSP-HER2ME can activate the HLA-A2+PBMC, mature the DCs derived from mononuclear cells, induce andgenerate the HER2 specific CTLs.